Gamma-aminobutyric acid uptake by rat kidney brush-border membrane vesicles

Summary Brush-border membrane vesicles (BBMV) from rat kidney cortex possessed two uptake systems for -aminobutyric acid (GABA), a high affinity system (Km=10.9 M) and a low affinity system (Km=1203 M). Both uptake systems were inhibited by p-hydroxymercuribenzoic acid and ouabain, and by the action of neuraminidase, whereas the GABA analogs nipecotic acid, -alanine, 2,4-diaminobutyric acid and 4,5,6,7-tetrahydroisoxazolo-[4,5c]-pyridin-3-ol had no effect on the GABA uptake activity. The BBMW uptake systems were clearly different from the GABA transport systems present in brain tissue.     

Inhibition of high affinity uptake of GABA by branched fatty acids

Summary Several branched fatty acids including an antiepileptic agent nDPA were tested as potential inhibitors of high affinity uptake of GABA by brain slices and synaptosomes. Only three compounds (2-butyl-3-propylhexanoic acid, 5-propyloctanoic acid, 2-propylpenten-2-oic acid) were found to be relatively weak inhibitors of the uptake system. There was no correlation between anticonvulsant properties of the branched fatty acids and their potencies as inhibitors of high affinity uptake of GABA.     

Reduction of high affinity glutamate uptake in rat hippocampus by two polyamine-like toxins isolated from the venom of the predatory wasp Philanthus triangulum F

Two components of the venom of the predatory wasp Philanthus triangulum F. significantly reduce--to a greater or less extent--the high affinity uptake of glutamate in rat hippocampus. A concentration of 10 microM delta-PTX caused a reduction of 74%, while the other component, beta-PTX, at the same concentration, caused a reduction of 18%. Hence the effect of delta-PTX on high affinity glutamate uptake in the hippocampus is comparable with its effect on high affinity glutamate uptake in insect neuromuscular junctions. Contrary to our previous findings that beta-PTX has no effect on high affinity glutamate uptake in insect glutamatergic terminal axons, however, beta-PTX significantly reduces high affinity glutamate uptake in the hippocampus, albeit less effectively than delta-PTX.     

Cl<Superscript>–</Superscript> affects the function of the GABA cotransporter rGAT1 but preserves the mutal relationship between transient and transport currents

The effects of reducing external Cl- on the electrophysiological properties of the Na+/Cl(-)-dependent GABA transporter rGAT1 expressed in Xenopus oocytes were investigated. In agreement with a recently proposed kinetic scheme, the effects of Cl- are complex but preserve the mutual relationship that links the transport-associated current, I(tr), measured in saturating GABA concentration, and the transient current, I(pre), recorded in the absence of GABA following a voltage step from the holding potential Vh to V. In particular, I(tr) (V) - I(tr) (Vh) = r integral I(pre) (V) dt, where r is the relaxation rate of I(pre) at the same membrane potential and Cl- concentration. The model also predicts a relationship between charge relaxation rate and apparent affinity for GABA, which is also verified in the presence of lowered Na+ or Cl- concentrations. In these conditions, the binding rate of GABA to the transporter is increased. All these effects are consistent with the hypothesis that interaction of the organic substrate with rGAT1 induces a conversion from a capacitive to a conductive mode of operation without strongly altering either the amount or the rate of charge movement.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

S-nitrosoglutathione reductase activity of human and yeast glutathione-dependent formaldehyde dehydrogenase and its nuclear and cytoplasmic localisation

S-nitrosoglutathione (GSNO) formation represents a mechanism for storage and transport of nitric oxide. Analysis of human liver and Saccharomyces cerevisiae extracts has revealed the presence of only one enzyme able to significantly reduce GSNO, identified as glutathione-dependent formaldehyde dehydrogenase (FALDH). GSNO is the best substrate known for the human and yeast enzymes (kcat/Km = 444,400 and 350,000 mM(-1) min(-1), respectively). Although NADH is the preferred cofactor, some activity with NADPH (Km = 460 microM) can be predicted in vivo. The subcellular localization demonstrates a cytosolic and nuclear distribution of FALDH in living yeast cells. This agrees with previous results in rat, and suggests a role in the regulation of GSNO levels in the cytoplasmic and nuclear compartments of the eukaryotic cell.     

Connection between 3H 5-HT and adenyl cyclase activation induced by 5-HT in preparations of cerebral glial membranes

Purified glial membrane preparations have been isolated from horse brain striatum. Tritiated 5-HT bound to these membranes with a high affinity (KD = 10 nM); the corresponding binding is reversible and appears specific of the serotoninergic structure. In parallel, 5-HT activates an adenylate cyclase with a low affinity (KD = 1 microM). The sites involved in this binding and in this adenylate cyclase activation appear different from the serotoninergic sites reported in the neuronal membrane preparations.     

Action ofβ-(4-chlorophenyl-GABA) on uptake and metabolism of GABA in different subcellular fractions of rat brain

Summary -(4-chlorophenyl)-GABA, a GABA mimetic compound, acts as an inhibitor of GABA metabolism in both synaptosomal and extrasynaptosomal compartments. It has no significant action on GABA or Glu uptake by synaptosomes.     

Effect of lipophilic cations on thiamine transport system in isolated rat hepatocytes

The effect of lipophilic cations such as triphenylmethylphosphonium, tetraphenylphosphonium and tetraphenylarsonium in addition to dibenzyldimethylammonium on thiamine transport in isolated rat hepatocytes was studied. Lipophilic cations at the concentration 10 microM almost completely inhibited thiamine uptake. Kinetic studies showed that these compounds were competitive inhibitors with a very high affinity. These results suggest that lipophilic cations in addition to quaternary ammonium compounds also share a common binding site for thiamine in isolated rat hepatocytes.     

Presence of specific binding sites for [3H]sarcophytol-A in cultured human skin fibroblasts

The presence of specific binding sites for [3H]sarcophytol-A in human skin fibroblasts was examined using biochemical and morphological methods. The displacement studies clearly revealed that high (KD = 31.0 nM) and low (KD = 6.05 microM) affinity sites were present in the intact cells. Moreover, autoradiographic studies using light microscopy revealed that the specific binding sites may exist in both the cytoplasm and the nuclei.     

Isoguvacine, isonipecotic acid, muscimol and N-methyl isoguvacine on the GABA receptor in rat sympathetic ganglia.

The GABA-mimetic activities of 4 analogues muscimol, isonipecotic acid, isoguvacine and N-methyl isoguvacine have been examined at the GABA receptor in the rat isolated superior cervical ganglion. The depolarizing action of all 4 analogues could be selectively antagonized by bicuculline methochloride and isopropyl bicyclophosphate. Muscimol was the only analogue more potent than GABA (molar potency ratio = 5.08 +/- 0.707). The potency of isoguvacine was 0.23 +/- 0.026 and isonipecotic acid 0.011 +/- 0.0028. N-methyl isoguvacine was less than 0.001 GABA.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Summary Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptornegative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [³H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10^–6 M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10^–5 M linoleic acid or 10^–5 M arachidonic acid but not by 10^–6 M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (K_d 0.54 nM±0.11 nM; n=6) than growth in medium supplemented with untreated serum (complete medium) (K_d=1.68 nM±0.48 nM; n=6) (p<0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10^–5 M linoleic acid or 10^–5 M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10^–5 M stearic acid or 10^–5 M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10^–5 M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [³H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [³H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Isoguvacine,isonipecotic acid,muscimol and N-methyl isoguvacine on the GABA receptor in rat sympathetic ganglia

Summary The GABA-mimetic activities of 4 analogues muscimol, isonipecotic acid, isoguvacine and N-methyl isoguvacine have been examined at the GABA receptor in the rat isolated superior cervical ganglion. The depolarizing action of all 4 analogues could be selectively antagonized by bicuculline methochloride and isopropyl bicyclophosphate. Muscimol was the only analogue more potent than GABA (molar potency ratio=5.08±0.707). The potency of isoguvacine was 0.23±0.026 and isonipecotic acid 0.011±0.0028. N.-methyl isoguvacine was <0.001 GABA.     

Human myeloperoxidase activity is inhibited in vitro by quercetin. Comparison with three related compounds

Quercetin is an effective inhibitor of human myeloperoxidase (MPO) activity, both with purified enzyme (IC50 = 3.5 microM) and in a system using stimulated human neutrophils. Quercetin is significantly more potent than three other related compounds (rutin, rutin sulfate and troxerutin) and than methimazole, a previously-known myeloperoxidase inhibitor. The inhibitory activity of quercetin is of the competitive type. Moreover, quercetin is directly able to scavenge hypochlorous acid (HOCl), a chlorinated species generated by the MPO/H2O2/Cl- system.     

The metabotropic GABA receptor: molecular insights and their functional consequences

Recent years have seen rapid and significant advances in our understanding of the G-protein-coupled gamma-amino butyric acid, B-type (GABA(B)) receptor, which could be a therapeutic target in conditions as diverse as epilepsy and hypertension. This progress originated with the ground-breaking work of Bernhard Bettler's team at Novartis who cloned the DNA encoding a GABA(B) receptor in 1997. Currently, the receptor is thought to be an unusual, possibly unique, example of a heterodimer composed of homologous, seven-transmembrane-domain (7TMD) subunits (named GABA(B) R1 and GABA(B) R2), neither of which is fully functional when expressed alone. The large N-terminal domain of the GABA(B) R1 subunit projects extracellularly and contains a ligand binding site. The similarity of the amino acid sequence of this region to some bacterial periplasmic amino acid-binding proteins of known structure has enabled structural and functional modelling of the N-terminal domain, and the identification of residues whose substitution modulates agonist/antagonist binding affinities. The intracellular C-terminal domains of the R1 and R2 subunits appear to constitute an important means of contact between the two subunits. Alternative splice variants, a common and functionally important feature of 7TMD proteins, have been demonstrated for the R1 subunit. Notably GABA(B) R1a differs from GABA(B) R1b by the possession of an N-terminal extension containing two complement protein modules (also called SCRs, or sushi domains) of unknown function. The levels at which each of the respective variants is expressed are not equal to one another, with variations occurring over the course of development and throughout the central nervous system. It is not yet clear, however, whether one variant is predominantly presynaptically located and the other postsynaptically located. The existence of as yet unidentified splice variants, additional receptor subtypes and alternative quaternary composition has not been ruled out as a source of receptor heterogeneity.     

Oxidation of synephrine by type A and type B monoamine oxidase.

Synephrine (SP) was found to be a substrate for monoamine oxidase (MAO) in rat brain mitochondria, showing the Km and Vmax values of 250 microM and 32.6 nmoles/mg of protein/30 min respectively. The inhibition studies showed that the SP oxidation was carried out by both type A and type B MAO and a major part of the activity was due to type A MAO.     

Uptake of3H-GABA (γ-aminobutyric acid) and3H-leucine in the pancreatic islets and substantia nigra of the rat

Summary Isolated pancreatic islets and thin slices of substantia nigra (SN) of the rat were incubated in a medium containing³H-GABA or³H-leucine to test the activity of both tissues in the uptake of those substances. Pancreatic islets showed a low uptake of both³H-GABA and³H-leucine, but SN had a high activity in the uptake of³H-GABA, though not for³H-leucine. This suggests that GABA contained at high levels in the pancreatic islets plays some functional role other than in neurotransmission as in the central nervous system (CNS).     

The action of conformationally restricted analogues of GABA onLimulus andHelix central neurones

Summary Intracellular recordings have been made from GABA sensitive neurones in the central nervous system ofLimulus andHelix. The following conformationally restricted analogues of GABA all possessed GABA-like activity onLimulus neurones andHelix excitatory GABA receptors: muscimol, thiomuscimol, THIP, isoguvacine and piperidine-4-carboxylic acid. It is suggested that GABA interacts with these receptors in a partially extended and almost planar conformation.     

Role of the conserved glutamine 291 in the rat γ-aminobutyric acid transporter rGAT-1

We investigated the role of the Q291 glutamine residue in the functioning of the rat γ-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wild-type and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhydryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter. Received 24 October 2005; accepted 11 November 2005     

Adenosine uptake in pyrimidine 5'-nucleotidase deficient human erythrocytes via a high affinity transport system

Using a pulse-labeling technique, 14C-adenosine uptake into pyrimidine 5'-nucleotidase (P5N) deficient erythrocytes (RBC) was found to be impaired. The Lineweaver-Burk plot showed Km values of 2.0 X 10(-3) mM and 0.2 X 10(-3) mM for normal RBC and P5N deficient RBC, respectively. These results indicate that P5N is one of regulators of the adenosine transport system and/or is associated with adenosine carrier protein.