Characterization of adenosine receptors in a model of cultured neurons from rat forebrain

The neuromodulator adenosine is acting through specific receptors coupled to adenylate cyclase via G-proteins. The expression of both adenosine receptors A₁ and A₂ as well as forkolin binding sites was investigated by radioligand binding techniques in 8-day-old neurons isolated from fetal rat forebrain and cultured in chemically-defined medium. Adenosine A₁ receptors were specifically labeled with [³H]chloro-N⁶-cyclopentyladenosine (CCPA), whereas [³H]CGS 21680 was used for the analysis of A₂ receptors. Cultured neurons exhibited high affinity binding sites for CCPA (Bmax=160 fmol/mg protein; Kd=2.9 nM), and for CGS 21680 (Bmax=14 fmol/mg protein; Kd=1.7 nM). These data correlate well with those obtained in crude membranes isolated from the newborn rat forebrain. The incubation of culture membranes in the additional presence of guanylyl-5-imidodiphosphate (Gpp(NH)p, a GTP analogue) led to significantly increased Kd-values, suggesting the association of adenosine receptors with G-proteins. Finally, cultured neurons also bound specifically [³H]forskolin with characteristics close to those found in the newborn brain, indicating that cultured neurons appear as an appropriate model for studying the neuromodulatory properties of adenosine.     

Effects of Linalool on Glutamate Release and Uptake in Mouse Cortical Synaptosomes

Linalool, a monoterpene compound prevalent in essential oil of plant species traditionally used as sedatives, has been characterized as anticonvulsant in several experimental models. Linalool inhibits the binding of [³H]glutamate and [³H]dizocilpine to brain cortical membranes, indicating a participation of the glutamatergic transmission its mechanism of action. In this study, we investigated the effects of linalool on [³H]glutamate release (basal and potassium-stimulated) and [³H]glutamate uptake in mice cortical synaptosomes. Linalool significantly reduced potassium-stimulated glutamate release as well as glutamate uptake, not interfering with basal glutamate release. The data indicates that linalool may interfere with several relevant elements of the glutamatergic transmission, including detriment of the K⁺-stimulated glutamate release.     

Age and Brain Structural Related Effects of Glutaric and 3-Hydroxyglutaric Acids on Glutamate Binding to Plasma Membranes During Rat Brain Development

(1) In the present study we determined the effects of glutaric (GA, 0.01–1 mM) and 3-hydroxyglutaric (3-OHGA, 1.0–100 μM) acids, the major metabolites accumulating in glutaric acidemia type I (GA I), on Na⁺-independent and Na⁺-dependent [³H]glutamate binding to synaptic plasma membranes from cerebral cortex and striatum of rats aged 7, 15 and 60 days. (2) GA selectively inhibited Na⁺-independent [³H]glutamate binding (binding to receptors) in cerebral cortex and striatum of rats aged 7 and 15 days, but not aged 60 days. In contrast, GA did not alter Na⁺-dependent glutamate binding (binding to transporters) to synaptic membranes from brain structures of rats at all studied ages. Furthermore, experiments using the glutamatergic antagonist CNQX indicated that GA probably binds to non-NMDA receptors. In addition, GA markedly inhibited [³H]kainate binding to synaptic plasma membranes in cerebral cortex of 15-day-old rats, indicating that this effect was probably directed towards kainate receptors. On the other hand, experiments performed with 3-OHGA revealed that this organic acid did not change Na⁺-independent [³H]glutamate binding to synaptic membranes from cerebral cortex and striatum of rats from all ages, but inhibited Na⁺-dependent [³H]glutamate binding to membranes in striatum of 7-day-old rats, but not in striatum of 15- and 60-day-old rats and in cerebral cortex of rats from all studied ages. We also provided some evidence that 3-OHGA competes with the glutamate transporter inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting a possible interaction of 3-OHGA with glutamate transporters on synaptic membranes. (3) These results indicate that glutamate binding to receptors and transporters can be inhibited by GA and 3-OHGA in cerebral cortex and striatum in a developmentally regulated manner. It is postulated that a disturbance of glutamatergic neurotransmission caused by the major metabolites accumulating in GA I at early development may possibly explain, at least in part, the window of vulnerability of striatum and cerebral cortex to injury in patients affected by this disorder.     

High-affinity binding ofl-glutamate to chick retinal membranes

Binding ofl-[³H]glutamate to membranes from whole chick retina and from subcellular fractions enriched with photoreceptor terminals (P₁), or terminals from the inner plexiform layer (P₂) was studied. Na⁺-dependent and Na⁺-independent binding to these membranes was demonstrated. Na⁺-independent binding was stereospecific. Kinetic analysis of the binding process indicated a single high-affinity system (K _B=0.55 M) with a capacity of approximately 20 pmoles/mg protein in all the membrane fractions. [³H]Glutamate binding to P₁ and P₂ fractions was effectively displaced by several structural analogues of glutamate. Glutamate diethyl-ester appreciably displaced binding, whereas kainic acid did not displace bound glutamate. Data indicate the binding of [³H]glutamate to physiologically relevant receptors in the chick retina.     

In vitro neuropharmacological evaluation of penitrem-induced tremorgenic syndromes: importance of the GABAergic system

The effects of the fungal neurotoxin penitrem A on the GABAergic and glutamatergic systems in rat brain were evaluated. Penitrem A inhibited binding of the GABA_A-receptor ligand [³H]TBOB to rat forebrain and cerebellar membrane preparations with IC₅₀ (half maximal inhibitory concentration) values of 11 and 9 μM, respectively. Furthermore, penitrem A caused a concentration-dependent increase of [³H]flunitrazepam and [³H]muscimol binding in rat forebrain, but not in cerebellar preparations. The stimulation of [³H]flunitrazepam binding by penitrem A was abolished by the addition of GABA. In cerebellar preparations, a different pharmacological profile was found, with penitrem A allosterically inhibiting [³H]TBOB binding by interacting with a bicuculline-sensitive site. Moreover, penitrem A inhibited the high affinity uptake of GABA and glutamate into cerebellar synaptosomes with IC₅₀ values of 20 and 47 μM, respectively. The toxin showed no effect on NMDA or AMPA glutamate receptor binding. In conclusion, our results suggest that penitrem A exerts region-specific effects in the brain, leading to positive modulation of GABA_A-receptor function in forebrain. Conversely, penitrem A may act as a bicuculline-like convulsant in cerebellum.     

[3H]Aniracetam Binds to Specific Recognition Sites in Brain Membranes

Abstract: [³H]Aniracetam bound to specific and saturable recognition sites in membranes prepared from discrete regions of rat brain. In crude membrane preparation from rat cerebral cortex, specific binding was Na⁺ independent, was still largely detectable at low temperature (4°C), and underwent rapid dissociation. Scatchard analysis of [³H]aniracetam binding revealed a single population of sites with an apparent K_D value of ～70 nM and a maximal density of 3.5 pmol/mg of protein. Specifically bound [³H]aniracetam was not displaced by various metabolites of aniracetam, nor by other pyrrolidinone-containing nootropic drugs such as piracetam or oxiracetam. Subcellular distribution studies showed that a high percentage of specific [³H]aniracetam binding was present in purified synaptosomes or mitochondria, whereas specific binding was low in the myelin fraction. The possibility that at least some [³H]aniracetam binding sites are associated with glutamate receptors is supported by the evidence that specific binding was abolished when membranes were preincubated at 37°C under fast shaking (a procedure that substantially reduced the amount of glutamate trapped in the membranes) and could be restored after addition of either glutamate or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) but not kainate. The action of AMPA was antagonized by DNQX, which also reduced specific [³H]aniracetam binding in unwashed membranes. High levels of [³H]aniracetam binding were detected in hippocampal, cortical, or cerebellar membranes, which contain a high density of excitatory amino acid receptors. Although synaptosomal aniracetam binding sites may well be associated with AMPA-sensitive glutamate receptors, specifically bound [³H]aniracetam could not be displaced by cyclothiazide or GYKI 52466, which act as a positive and negative modulator of AMPA receptors, respectively.     

Effects of amphipathic drugs onl-[3H]glutamate binding to synaptic membranes and the purified binding protein

Four amphipathic molecules with known local anesthetic activity, dibucaine, tetracaine, chlorpomazine, and quinacrine, inhibited the binding ofl-[³H]glutamic acid to rat brain synaptic plasma membranes and to the purified glutamate binding protein. Neither haloperidol nor diphenylhydantoin had significant inhibitory effects on the glutamate binding activity of the membranes or of the purified protein. The amphipathic drugs apparently inhibitedl-[³H]glutamate binding to synaptic membranes by a mixed type of inhibition. The inhibitory activity of quinacrine on glutamate binding to the synaptic membranes was greater in a low ionic strength, Ca²⁺-free buffer medium, than in a physiologic medium (Krebs-Henseleit buffer). Removal of Ca²⁺ from the Krebs solution enhanced quinacrine's inhibition of glutamate binding. Quinacrine up to 1 mM concentration did not inhibit the high affinity Na⁺-dependentl-glutamate transport in these membrane preparations. The importance of Ca²⁺ in the expression of quinacrine's effects on the glutamate binding activity of synaptic membranes and the observed tetracaine and chlorpromazine-induced increases in the transition temperature for the glutamate binding process of these membranes, were indicative of an interaction of the local anesthetics with the lipid environment of the glutamate binding sites.     

Comparison of Effects of DL-Threo-β-Benzyloxyaspartate (DL-TBOA) and L-Trans-Pyrrolidine-2,4-Dicarboxylate (t-2,4-PDC) on Uptake and Release of [3H]D-Aspartate in Astrocytes and Glutamatergic Neurons

Uptake and release processes in cerebellar astrocytes and granule neurons (glutamatergic) for glutamate were investigated by the use of [³H]D-aspartate, a non-metabolizable glutamate analog. The effects of DL-threo--benzyloxyaspartate (DL-TBOA) and L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC) on uptake and release of [³H]D-aspartate were studied. Both compounds inhibited potently uptake of [³H]D-aspartate in neurons and astrocytes (IC₅₀ values 10-100 M), DL-TBOA being slightly more potent than t-2,4-PDC. Release of preloaded [³H]D-aspartate from neurons or astrocytes could be stimulated by addition of excess t-2,4-PDC whereas addition of DL-TBOA had no effect on [³H]D-aspartate efflux. Moreover, DL-TBOA inhibited significantly the depolarization-induced (55 mM KCl) release of preloaded [³H]D-aspartate in the neurons. The results reflect the fact that DL-TBOA is not transported by the glutamate carriers while t-2,4-PDC is a substrate which may heteroexchange with [³H]D-aspartate. It is suggested that DL-TBOA may be used to selectively inhibit depolarization coupled glutamate release mediated by reversal of the carriers.     

Molecular characteristics of glutamate receptors in the mammalian brain

Summary The strong excitatory activity of L-glutamic acid on central nervous system neurons is thought to be produced by interaction of this amino acid with specific neuronal plasma membrane receptors. The binding of L-glutamate to these surface receptors brings about an increase in membrane permeability to Na⁺ and Ca²⁺ ions presumably through direct activation of ion channels linked to the membrane receptors. The studies described in this paper represent attempts to define the subcellular distribution and pharmacological properties of the recognition site for L-glutamic acid in brain neuronal preparations, to isolate and explore the molecular characteristics of the receptor recognition site, and, finally, to demonstrate the activation of Na⁺ channels in synaptic membranes following the interaction of glutamate with its receptors.Radioligand binding assays with L-[³H] glutamic acid have been used to demonstrate a relative enrichment of these glutamate recognition sites in isolated synaptic plasma membranes. The specific binding of L-[³H] glutamate to these membrane sites exhibits rapid association and dissociation kinetics and rather complex equilibrium binding kinetics. The glutamate binding macromolecule from synaptic membranes has been solubilized and purified and was shown to be a small molecular weight glycoprotein (M_T 13 000). This protein tends to form aggregates which have higher specific activity at low concentrations of glutamate than the M_T 13 000 protein has. The overall affinity of the purified protein is lower than that of the high affinity sites in the membrane. Nevertheless, the purified protein exhibits pharmacological characteristics very similar to those of the membrane binding sites. On the basis of its pharmacological properties this protein belongs in the category of the physiologic glutamate preferring receptors.By means of differential solubilization of membrane proteins with Na-cholate, it was shown that this recognition site is an intrinsic synaptic membrane protein whose binding activity is enhanced rather than diminished by cholate extraction of the synaptic membranes. The role of membrane constituents in regulating the binding activity of this protein has been explored and a possible modulation of glutamate binding by membrane gangliosides has been demonstrated. Finally, this glutamate binding glycoprotein is a metalloprotein whose activity is dependent on the integrity of its metallic (Fe) center. This is a clear distinguishing characteristic of this protein vis-à-vis the glutamate transport carriers.The presence of functional glutamate receptors in synaptosomes and resealed synaptic plasma membranes has also been documented by the demonstration of glutamate-activated Na⁺ flux across the membrane of these preparations. The bidirectionality, temperature independence, and apparent desensitization of this stimulated flux following exposure to high concentrations of glutamate are properties indicative of a receptor-initiated ion channel activation. It would appear, then, that the synaptic membrane preparations provide a very useful system for the study of both recognition and effector function of the glutamate receptor complex.     

Uptake and metabolism of l-[3H]pyroglutamic acid in neuronal and glial cells in culture

The presence of an efficient uptake system for l-pyroglutamate was demonstrated in cultured glial cells originating from newborn rats. This compound is also transported by a high affinity uptake mechanism in neurons cultured from rat embryos cerebral hemispheres, but the V_max is 6 times lower than for glial cells. It is shown that l-pyroglutamate like l-glutamate is preferentially transported by glial cells, but with a V_max 40 to 60 times lower than for glutamate. The metabolism of l-pyroglutamate was also studied in cultured rat neuronal and glial cells, using l-[³H]pyroglutamate. Pyroglutamate, its metabolites and the various amino acids were separated by thin-layer electrophoresis. [³H]Pyroglutamate is more actively metabolised in glial cells than in neurons and glutamate is the main metabolite. Glutamate maximal specific activity is 4 times higher in glial than in neuronal cultures. It should also be noted that some [³H]pyroglutamate is transformed in [³H]GABA after longer incubation periods, but only in neurons. These results show the importance of glial cells for pyroglutamate uptake and metabolism in nervous tissue. They also suggest that pyroglutamate may interfere with glutamate neurotransmission in vivo.     

Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

[³H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K⁺. By utilising high K⁺ incubations to estimate non-specific [³H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 · 10^?7 M and the total maximum binding to be 2.33 · 10⁵ sites/cell. Ouabain inhibition of (Na⁺, K⁺)-pump function was monitored by the cellular uptake of ^B6Rb over 5 min. The larger fraction of ^B6Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 · 10^?7 M. The cellular distribution of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was investigated using [³H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [³H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.     

[3H]cocaine labels a binding site associated with the serotonin transporter in guinea pig brain: Allosteric modulation by paroxetine

We studied the characteristics of [³H]cocaine binding to membranes prepared from whole guinea pig brain. Cocaine binding was specific and saturable. A one-site binding model fit the data adequately: the Kd value of [³H]cocaine was 44 nM with a Bmax value of 280 fmol/mg protein. The rank order of potency for the [³H]cocaine binding site was paroxetine > clomipramine > (–)-cocaine > fluoxetine > mazindol > desipramine > GBR12909 > phencyclidine > benztropine > GBR12935 > (+)-cocaine. The IC50 values of these drugs for inhibition of [³H]cocaine binding were highly correlated with their IC50 values for inhibition of [³H]5-HT uptake into synaptosomes prepared from whole guinea pig brain. High affinity 5-HT uptake inhibitors produced dose-dependent wash-resistant (pseudoirreversible) inhibition of [³H]cocaine binding. The wash-resistant inhibition produced by paroxetine was due to an increase in the Kd of [³H]cocaine binding sites, and was accompanied by an increase in the dissociation rate, consistent with an allosteric mechanism. These studies suggest that, using membranes prepared from whole guinea pig brain, [³H]cocaine labels a binding site associated with serotonin transporter and that paroxetine and cocaine bind to different sites on the serotonin transporter.Abbreviations GBR12909 1-(2-{bis(4-fluorophenyl)methoxy}ethyl)-4-{3-phenylpropyl}piperazine - TCP 1-{1-(2-thienyl)cyclohexyl}piperidine - BTCP N-{1-(2-benzo(b)thiophenyl)cyclohexyl}piperidine - PCP 1-(1-phenylcyclohexyl)piperidine - GBR12935 (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine) - CMI clomipramine     

Differential effects of metal ligands on synaptic membrane glutamate binding and uptake systems

The high affinity, Na⁺-independentl-[³H]glutamate binding process in synaptic membranes and in the purified binding protein was shown to be inhibited to an almost equal extent by the metal ligands NaN₃, KCN, ando-phenanthroline, and by 2,4,5-trihydroxyphenylalanine (6-OH DOPA). The high affinity, Na⁺-dependent glutamate transport activity in these membranes was almost totally insensitive to NaN₃,o-phenanthroline, KCN, and 6-OH DOPA. These agents, especially 6-OH DOPA, may be useful tools in achieving a discrimination between putative physiologic receptors and uptake carrier sites forl-glutamate in synaptic membranes. The sensitivity of the glutamate binding sites to the effects of the metal ligands may be correlated to the presence of an iron-sulfur center in the purified glutamate binding protein. Some of the characteristics of this metallic center were explored by optical and paramagnetic resonance spectroscopic techniques and are described in this study.This research was supported by grants DAAG29-79-C-0156 from the Army Research Office and AA 04732 from NIAAA.     

Interference of S-Alkyl Derivatives of Glutathione with Brain Ionotropic Glutamate Receptors

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on ⁴⁵Ca²⁺ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na⁺-independent binding of L-[³H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[³H]methyl-4-isoxazolepropionate [³H]AMPA (IC₅₀ values: 0.8–15.9 M). S-Alkylation of glutathione rendered the derivatives unable to inhibit [³H]kainate binding. The NMDA-sensitive binding of L-[³H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-³H]propyl-1-phosphonate ([³H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [³H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [³H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of ⁴⁵Ca²⁺ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their -glutamyl moieties.     

Exposure to Ebselen Changes Glutamate Uptake and Release by Rat Brain Synaptosomes

We investigated effects of Ebselen, diphenyl diselenide (PhSe)₂ and diphenyl ditelluride (PhTe)₂ on [³H]glutamate uptake and release by brain synaptosomes. Ebselen after acute exposure inhibited K⁺-stimulated [³H]glutamate release by brain synaptosomes. (PhSe)₂ and (PhTe)₂ did not change [³H]glutamate release by brain synaptosomes. Ebselen, (PhSe)₂ and (PhTe)₂ had no significantly effects on [³H]glutamate uptake after acute exposure. In vitro, Ebselen (100 M) inhibited [³H]glutamate release and uptake. (PhSe)₂ had no significant effect, while (PhTe)₂ (100 M) inhibited [³H]glutamate uptake by brain synaptosomes. In vitro, (PhSe)₂, (PhTe)₂ and Ebselen caused a significant inhibition of [³H]glutamate uptake by brain synaptic vesicles in vitro. The results demonstrated that organochalcogenides have a rather complex effect on glutamate homeostasis depending on the compound and the schedule of exposition. We propose that the neuroprotective action of Ebselen can be related, in addition to its glutathione peroxidase-like and antilipoperoxidative activity, to a direct interaction with the glutamatergic system by reducing K^ï-evoked glutamate release.     

Kainic acid binding to membranes of striatal neurons.

The specific binding of [³H]-kainic acid to membrane fragments of rat striatum was examined. The specific binding was found to be saturable and of high affinity. The dissociation constant was about 71 nM, while the apparent maximal number of receptor sites was 254 fmoles/mg protein. [³H]-Kainic acid binding was effectively competed by both unlabeled kainic acid and glutamate, Lesions of the striatum by stereotaxic injection of 5 nmoles of kainic acid reduced the density of [³H]-kainic acid binding sites by half, without affecting their affinity. Lesions of the cortico-striatal afferents, however, did not affect the binding of [³H]-kainic acid, although sodium-dependent glutamate uptake was reduced by 30%. It is concluded that [³H]-kainic acid binds to a population of receptors localized on neurons of the caudate-putamen.     

Binding Sites for [3H]Glutamate and [3H]Aspartate in Human Cerebellum

Abstract The binding of [³H]aspartate and [³H]glutamate to membranes prepared from frozen human cerebellar cortex was studied. The binding sites differed in their relative proportions, their inhibition by amino acids and analogues, and by the effects of cations. A proportion (about 30%) of [³H]glutamate binding was to sites similar to those labelled by [³H]aspartate. An additional component of [³H]gluta-mate binding (about 50%) was displaced by quisqualate and aL-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and may represent a “quisqualate-preferring” receptor. Neither N-methyl-d-aspartic acid-sensitive nor dl-2-amino-4-phosphonobutyric acid-sensitive [³H]glutamate binding was detected.     

Glutamate receptor changes in brain synaptic membranes from human alcoholics

Brains from human alcoholics and non-alcoholics were obtained shortly after death. The hippocampus was dissected, homogenized, and processed for the isolation of a synaptic membraneenriched fraction and the study ofl-[³H]glutamic acid and 3-((±)-2-carboxypiperazin-4-yl)-[1,2³H]propyl-l-phosphonic acid ([³H]CPP) binding sites. The pharmacological characteristics ofl-[³H]glutamic acid binding to synaptic membranes isolated from hippocampus corresponded to the labeling of a mixture of N-methyl-d-aspartate (NMDA), kainate and quisqualic acid receptor sites. Synaptic membranes prepared from the hippocampus of individuals classified as alcoholics had significantly higher density of glutamate binding sites than identically prepared membranes from non-alcoholic individuals. In addition, there was a clear definition of a population ofl-glutamate binding sites (approx. 10% of total) in the membranes from alcoholics that had a higher affinity for the ligand than the major set of sites labeled in membranes from both alcoholics and non-alcoholics. Neither the age of the individuals at the time of death nor the time that elapsed between death and processing of brain tissue were significant factors in determining either recovery of purified synaptic membranes from brain homogenates orl-[³H]glutamate binding to synaptic membranes. In order to determine whether some of the changes inl-[³H]glutamic acid binding were due to alterations in binding at the NMDA receptor subtype, we also measured binding of [³H]CPP to extensively washed crude synaptosomal membranes. Membranes from brains of alcoholics had higher affinity (3-fold) for [³H]CPP but lower binding capacity (3-fold) when compared with those of non-alcoholics. These observations suggest selective changes among different glutamate receptor subtypes in human brain under conditions of chronic alcohol intake.     

Targeting of auxin carriers to the plasma membrane: effects of monensin on transmembrane auxin transport in Cucurbita pepo L. tissue

Treatment of etiolated zucchini (Cucurbita pepo L.) hypocotyl tissue with sub-micromolar concentrations of the cationophore monensin rapidly (<20 min) inhibited the transport catalytic activity of the specific auxin-anion efflux carrier and reduced the inhibition of this carrier by the phytotropin N-1-naphthylphthalamic acid (NPA). Monensin inhibited the basipetal polar transport of indol-3yl-acetic acid (IAA) in long (30 mm) zucchini segments. At concentrations lower than 10^–5 mol·dm^–3 monensin did not affect uptake of the pH probe [2-¹⁴C]5,5-dimethyloxazolidine-2,4-dione (DMO) or that of the membrane-potential probe tetra[¹⁴C-phenyl]phosphonium bromide (TPP⁺), did not affect the response of IAA net uptake to external Ca²⁺ concentration and did not alter the metabolism of IAA. It was concluded that low concentrations of monensin inhibit transport through the Golgi apparatus of auxin efflux carrier protein and that the efflux carriers turn over very rapidly in the plasma membrane. Monensin pretreatment did not affect the saturable binding of [³H]NPA to microsomal membranes, indicating that the auxin-efflux catalytic sites and the NPA-binding sites are located on separate proteins. At higher concentrations (10^–5 mol·dm^–3) monensin inhibited both mediated uptake and mediated efflux components of IAA transport. This effect was at least in part attributable to perturbation by monensin of the driving forces for mediated uptake since high concentrations of monensin also reduced the uptake of DMO and TPP⁺.Abbreviations CH cycloheximide - DMO 5,5-dimethyloxazolidine-2,4-dione - MDMP 2-(4-methyl-2,6-dinitroanlilino)N-methyl-propionamide - NPA N-1-naphthylphthalamic acid - TPP⁺ tetraphenylphosphonium ion We thank Mrs. R.P. Bell for technical assistance and Drs. G.F. Katekar and M.A. Venis for generous gifts of NPA. S.W. was supported by the U.K. Science and Engineering Research Council.     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.