(3)H](2S,4R)-4-Methylglutamate: a novel ligand for the characterization of glutamate transporters

[(3)H](2S,4R)-4-Methylglutamate ([(3)H]4MG), used previously as a ligand for low-affinity kainate receptors, was employed to establish a binding assay for glutamate transporters (GluTs), as 4MG has also been shown to have affinity for the glial GluTs, GLT1 and GLAST. In rat brain membrane homogenates in the presence of Na(+) ions at 4 degrees C, specific binding of [(3)H]4MG was rapid and saturable (t(1/2) approximately 15 min), representing > 90% of total binding. Dissociation of [(3)H]4MG occurred in a biphasic manner, however, saturation studies and Scatchard analysis indicated a single site of binding (n(H) = 0.85) and a K(d) of 6.2 +/- 0.8 microM with a B(max) of 111.8 +/- 23.8 pmol/mg protein. Specific binding of [(3)H]4MG was Na(+)-dependent and inhibited by K(+) and HCO(3-). Pharmacological inhibition with compounds acting at GluTs revealed that Glu, D- and L-aspartate, L-serine-O-sulfate and Ltrans-pyrrolidine-2,4-dicarboxylate fully displaced specific binding. Drugs having preferential affinity for GLT1, kainate, dihydrokainate and Lthreo-3-methylglutamate, all inhibited approximately 40% of specific binding. The inhibition pattern of L-serine-O-sulfate in the presence of a saturating concentration of dihydrokainate was suggestive of [(3)H]4MG also labelling GLAST. 6-Cyano-7-nitroquinoxaline, a kainate receptor antagonist, and a range of Glu receptor agonists and antagonists failed to significantly inhibit [(3)H]4MG binding. The pharmacological profile of binding of [(3)H]4MG resembled that found for [(3)H]D-aspartate, a ligand specific for GluTs, reinforcing the hypothesis that [(3)H]4MG was labelling GluTs in this assay. Together, these data illustrate the development of an efficient, economic binding assay that is suitable for the characterization of different subtypes of GLuTs.     

Pharmacological characterization of threo-3-methylglutamic acid with excitatory amino acid transporters in native and recombinant systems

The glutamate analog (+/-) threo-3-methylglutamate (T3MG) has recently been reported to inhibit the EAAT2 but not EAAT1 subtype of high-affinity, Na(+)-dependent excitatory amino acid transporter (EAAT). We have examined the effects of T3MG on glutamate-elicited currents mediated by EAATs 1-4 expressed in Xenopus oocytes and on the transport of radiolabeled substrate in mammalian cell lines expressing EAATs 1-3. T3MG was found to be an inhibitor of EAAT2 and EAAT4 but a weak inhibitor of EAAT1 and EAAT3. T3MG competitively inhibited uptake of D-[(3)H]-aspartate into both cortical and cerebellar synaptosomes with a similar potency, consistent with its inhibitory activity on the cloned EAAT2 and EAAT4 subtypes. In addition, T3MG produced substrate-like currents in oocytes expressing EAAT4 but not EAAT2. However, T3MG was unable to elicit heteroexchange of preloaded D-[(3)H]-aspartate in cerebellar synaptosomes, inconsistent with the behavior of a substrate inhibitor. Finally, T3MG acts as a poor ionotropic glutamate receptor agonist in cultured hippocampal neurons: concentrations greater than 100 microM T3MG were required to elicit significant NMDA receptor-mediated currents. Thus, T3MG represents a pharmacological tool for the study of not only the predominant EAAT2 subtype but also the EAAT4 subtype highly expressed in cerebellum.     

Resolution, absolute stereochemistry, and enantiopharmacology of the GluR1-4 and GluR5 antagonist 2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid

The phosphono amino acid, (RS)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl+ ++]propio nic acid (ATPO), is a structural hybrid between the NMDA antagonist (RS)-2-amino-7-phosphonoheptanoic acid (AP7) and the AMPA and GluR5 agonist, (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA). ATPO has been resolved into (S)-ATPO and (R)-ATPO using chiral HPLC, and the absolute stereochemistry of the two enantiomers was established by an X-ray crystallographic analysis of (R)-ATPO. (S)-ATPO and (R)-ATPO were characterized pharmacologically using rat brain membrane binding and electrophysiologically using the cortical wedge preparation as well as homo- or heteromeric GluR1-4, GluR5-6, and KA2 receptors expressed in Xenopus oocytes. (R)-ATPO was essentially inactive as an agonist or antagonist in all test systems. (S)-ATPO was an inhibitor of the binding of [(3)H]AMPA (IC(50) = 16 +/- 1 microM) and of [(3)H]-6-cyano-7-nitroquinoxaline-2,3-dione ([(3)H]CNQX) (IC(50) = 1.8 +/- 0.2 microM), but was inactive in the [(3)H]kainic acid and the [(3)H]-(RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([(3)H]CPP) binding assays. (S)-ATPO did not show detectable agonist effects at any of the receptors under study, but antagonized AMPA-induced depolarization in the cortical wedge preparation (IC(50) = 15 +/- 1 microM). (S)-ATPO also blocked kainic acid agonist effects at GluR1 (K(i) = 2.0 microM), GluR1+2 (K(i) = 3.6 microM), GluR3 (K(i) = 3.6 microM), GluR4 (K(i) = 6.7 microM), and GluR5 (K(i) = 23 microM), but was inactive at GluR6 and GluR6+KA2. Thus, although ATPO is a structural analog of AP7 neither (S)-ATPO nor (R)-ATPO are recognized by NMDA receptor sites.     

Identification of nuclear type II [(3)H]estradiol binding sites as histone H4

[3H]Luteolin binds covalently to uterine nuclear type II sites [B. Markaverich, K. Shoulars, M.A. Alejandro, T. Brown, Steroids 66 (2001) 707] and was used to identify this protein(s). SDS-PAGE analyses of [3H]luteolin-labeled type II site preparations revealed specific binding to 11- and 35-kDa proteins. The 11-kDa protein was identified as histone H4 by amino acid sequencing. Western blotting confirmed that the 11- and 35-kDa proteins were acetylated forms of histone H4. Anti-histone H4 antibodies (but not H2A, H2B, or H3 antibodies) quantitatively immunoadsorbed type II binding sites from nuclear extracts. Binding analyses by [3H]estradiol exchange, using luteolin as a competitor, detected specific type II binding activity to histone H4 (but not histones H2A, H2B, or H3) generated in a rabbit reticulocyte lysate translation system and confirmed that histone H4 is the type II site.     

Characterization of [(3)H]Quisqualate binding to recombinant rat metabotropic glutamate 1a and 5a receptors and to rat and human brain sections

We have investigated the binding properties of [(3)H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K:(D) values of 27 +/- 4 and 81 +/- 22 nM: for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S:)-4-Carboxyphenylglycine, (S:)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [(3)H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM: [(3)H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 microM: kainate, revealing the distribution of mGlu1+5 receptors. The latter were almost completely inhibited by the group I agonist (S:)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively.     

Effects of chronic antidepressant treatment on dopamine-related [3H]SCH 23390 and [3H]Spiperone binding in the rat striatum

1. The effects of chronic administration of antidepressants on dopamine-related [3H]SCH 23390 and [3H]spiperone binding to rat striatal membranes were assessed. 2. The monoamine oxidase inhibitors phenelzine (5 or 10 mg kg-1/day) and tranylcypromine (1 mg kg-1/day) and the tricyclic desipramine (10 mg kg-1/day) were administered for 28 days by constant subcutaneous infusion using Alzet (2ML4) osmotic minipumps. 3. These treatments did not alter Kd estimates for either [3H]SCH 23390 or [3H]spiperone binding sites. The monoamine oxidase inhibitors induced a decrease in the Bmax values for both [3H]SCH 23990 and [3H]spiperone binding sites. Desipramine induced a decrease in the Bmax value for [3H]SCH 23390 binding but had no effect on the Bmax value for [3H]spiperone binding.     

In vivo labeling of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone

Defects in mitochondrial energy metabolism have been implicated in several neurodegenerative disorders. Defective complex I (NADH:ubiquinone oxidoreductase) activity plays a key role in Leber's hereditary optic neuropathy and, possibly, Parkinson's disease, but there is no way to assess this enzyme in the living brain. We previously described an in vitro quantitative autoradiographic assay using [(3)H]dihydrorotenone ([(3)H]DHR) binding to complex I. We have now developed an in vivo autoradiographic assay for complex I using [(3)H]DHR binding after intravenous administration. In vivo [(3)H]DHR binding was regionally heterogeneous, and brain uptake was rapid. Binding was enriched in neurons compared with glia, and white matter had the lowest levels of binding. In vivo [(3)H]DHR binding was markedly reduced by local and systemic infusion of rotenone and was enhanced by local NADH administration. There was an excellent correlation between regional levels of in vivo [(3)H]DHR binding and the in vitro activities of complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase), suggesting that the stoichiometry of these components of the electron transport chain is relatively constant across brain regions. The ability to assay complex I in vivo should provide a valuable tool to investigate the status of this mitochondrial enzyme in the living brain and suggests potential imaging techniques for complex I in humans.     

Histamine H(2) -like receptors in chick cerebral cortex: effects on cyclic AMP synthesis and characterization by [(3) H]tiotidine binding

In this study, histamine (HA) receptors in chick cerebral cortex were characterized using two approaches: (1) analysis of the effects of HA-ergic drugs on the cAMP-generating system, and (2) radioreceptor binding of [(3) H]tiotidine, a selective H(2) antagonist. HA was a weak activator of adenylyl cyclase in a crude membrane preparation of chick cerebrum. On the other hand, HA (0.1-1000 microm) potently and concentration dependently stimulated cAMP production in [(3) H]adenine pre-labelled slices of chick cerebral cortex, displaying an EC(50) value (concentration that produces 50% of maximum response) of 2.65 microm. The effect of HA was mimicked by agonists of HA receptors with the following rank order of potency: HA >or= 4-methylHA (H(2)) >or= N alpha,N alpha-dimethylHA (H(3) > H(2) = H(1)) > 2-methylHA (H(1)) > 2-thiazolylethylamine (H(1)) >or= R alpha-methylHA (H(3)) > amthamine, dimaprit (H(2)), immepip (H(3), H(4)). The HA-evoked increase in cAMP production in chick cerebral cortex was antagonized by selective H(2) receptor blockers (aminopotentidine >or= tiotidine > ranitidine > zolantidine), and not significantly affected by mepyramine and thioperamide, selective H(1) and H(3) /H(4) receptor blockers, respectively. A detailed analysis of the antagonistic action of aminopotentidine (vs. HA) revealed a non-competitive mode of action. The binding of [(3) H]tiotidine to chick cortical membranes was rapid, stable and reversible. Saturation analysis resulted in a linear Scatchard plot, suggesting binding to a single class of receptor binding site with high affinity [equilibrium dissociation constant (K (d)) = 4.42 nm] and high capacity [maximum number of binding sites (B (max) ) = 362 fmol/mg protein]. The relative rank order of HA-ergic drugs to inhibit [(3) H]tiotidine binding to chick cerebrum was: antagonists - tiotidine > aminopotentidine = ranitidine >or= zolantadine > thioperamide - triprolidine; agonists - HA >or= 4-methylHA > 2-methylHA >or=R alpha-methylHA - dimaprit. In conclusion, chick cerebral cortex contains H(2) -like HA receptors that are linked to the cAMP-generating system and are labelled with [(3) H]tiotidine. The pharmacological profile of these receptors is different from that described for their mammalian counterpart. It is suggested that the studied receptors represent either an avian-specific H(2) -like HA receptors or a novel subtype of HA receptors.     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [H]8-OH-DPAT and other serotonergic ligands

[³H]8-OH-DPAT is a selective ligand for labeling 5-HT_1A receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [³H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [¹²⁵I]RTI-55 and [³H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [³H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [³H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [¹²⁵I]cyanopindolol, [³H]ketanserin/[³H]mesulergine, [³H]GR-65630, [³H]GR-113808 and [³H]LSD) that specifically labeled 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄ and 5-HT_5–7 receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Effects of Valeriana Officinalis Extracts on [3H]Flunitrazepam Binding,Synaptosomal [3H]GABA Uptake,and Hippocampal [3H]GABA Release

Extracts of Valeriana officinalis have been used in folkloric medicine for its sedative, hypnotic, tranquilizer and anticonvulsant effects, and may interact with -aminobutyric acid (GABA) and/or benzodiazepine sites. At low concentrations, valerian extracts enhance [³H]flunitrazepam binding (EC₅₀ 4.13 × 10^–10 mg/ml). However, this increased [³H]flunitrazepam binding is replaced by an inhibition at higher concentrations (IC₅₀ of 4.82 × 10^–1 mg/ml). These results are consistent with the presence of at least two different biological activities interacting with [³H]flunitrazepam binding sites. Valerian extracts also potentiate K⁺ or veratridine-stimulated release of radioactivity from hippocampal slices preloaded with [³H]GABA. Finally, inhibition of synaptosomal [³H]GABA uptake by valerian extracts also displays a biphasic interaction with guvacine. The results confirm that valerian extracts have effects on GABA_A receptors, but can also interact at other presynaptic components of GABAergic neurons.     

Characterization of 5-HT transporter and receptor system in HeLaS3 cells by [(3)H]8-OH-DPAT and other serotonergic ligands

[(3)H]8-OH-DPAT is a selective ligand for labeling 5-HT(1A) receptor sites. In competition binding experiments, we found that classic biogenic amine transporter inhibitors displaced [(3)H]8-OH-DPAT binding at its high-affinity binding sites in HeLaS3 cells. [(125)I]RTI-55 and [(3)H]paroxetine are known to specifically label amine transporter sites, and this was observed in our cells. Displacement studies showed that 8-OH-DPAT displayed affinity in a dose-dependent manner for the labeled amine transporter sites. These data suggest that [(3)H]8-OH-DPAT binds to amine uptake sites in HeLaS3 cells. A variety of drugs targeting different classes of receptors did not significantly affect [(3)H]8-OH-DPAT binding. Moreover, we determined the specific binding effects of various serotonergic ligands (i.e. [(125)I]cyanopindolol, [(3)H]ketanserin/[(3)H]mesulergine, [(3)H]GR-65630, [(3)H]GR-113808 and [(3)H]LSD) that specifically labeled 5-HT(1), 5-HT(2), 5-HT(3), 5-HT(4) and 5-HT(5-7) receptors, respectively. It is suggested that HeLaS3 cells contain distinct types of the related to 5-HT receptor recognition binding sites. These observations could help elucidate the relevant characteristics of different types of 5-HT receptors and 5-HT membrane transporters in tumor cells and their role in tumorigenesis.     

Binding of 3-hydroxybenzo[a]pyrene to bovine hemoglobin and albumin

Previous studies examined the bioavailability and first-pass biotransformation of 3-hydroxy[(3)H]benzo[a]pyrene ([(3)H]-3-OHBaP) in an isolated perfused catfish intestinal model. This work showed that 3-OHBaP, or a metabolite formed in intestine, bound covalently to blood protein. In this study, the blood adducts were characterized in vitro by incubating bovine ferric hemoglobin or albumin with [(3)H]-3OHBaP under various conditions. Incubation of 2 microM [(3)H]-3-OHBaP with hemoglobin for 1 h resulted in 7.49 pmol bound/mg protein, while albumin binding was 1.37 pmol/mg protein. Mild acid hydrolysis released only 5% of the radioactivity from 3-OHBaP-hemoglobin adducts. After gel filtration, the 3-OHBaP-hemoglobin adducts were examined by HPLC analysis. A single peak of radioactivity was detected at the same retention time as the heme component of hemoglobin. Unbound 3-OHBaP was oxidized to BaP-3,6-dione during incubation with ferric hemoglobin. Treatment of hemoglobin with ascorbic acid decreased the formation of hemoglobin adducts by 33%, while hydrogen peroxide treatment increased adduct formation by 44%. Incubation of [(3)H]-BaP-3-beta-D-glucuronide (BaP-3G) with hemoglobin and beta-glucuronidase resulted in greater binding to hemoglobin than incubation with [(3)H]-3-OHBaP alone. The hemoglobin adduct obtained from [(3)H]-BaP-3G also co-migrated with heme. These results indicate that an oxidative process is involved in formation of the heme adduct and that 3-OHBaP or BaP-3G might be a precursor of the bound metabolite.     

[3H]N-[1-(2-Thienyl)cyclohexyl]-3,4-Piperidine ([3H]TCP) Binding in Human Frontal Cortex: Decreases in Alzheimer-Type Dementia

We studied [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine [( 3H]TCP) binding to human frontal cortex obtained at autopsy from 10 histologically normal controls and eight histopathologically verified cases with Alzheimer-type dementia (ATD). Extensively washed membrane preparations were used to minimize the effects of endogenous substances. In ATD frontal cortex, the total concentration (Bmax) of [3H]TCP binding sites was significantly reduced by 40-50%. The apparent dissociation constant (KD) values showed no significant change. The reduction in binding capacity was also apparent in Triton X-100-treated membrane preparations, and there was a linear correlation between the number of [3H]TCP binding sites and that of N-methyl-D-aspartate (NMDA)-sensitive [3H]glutamate binding sites. [3H]TCP binding sites spared in ATD brains retained the affinity for the ligand and the reactivity to NMDA, L-glutamate, and glycine. These results suggest that the primary change in NMDA receptor-ion channel complex in ATD brains is the reduction of its number, possibly reflecting the loss of neurons bearing these receptor complexes, and that the functional linkage within the receptor complexes spared in ATD brains remains normal.     

Effects of bicuculline on [3H]SR 95531 binding in discrete regions of rat brains

Effects of bicuculline in vitro, and acute and chronic treatment of a subconvulsive dose of bicuculline on [³H]SR 95531 binding to discrete regions of rat brains were studied in Sprague-Dawley rats. Scatchard analysis of the binding isotherms exhibited two populations of binding sites for [³H]SR 95531 in frontal cortex, cerebellum, striatum and substantia nigra. The apparent K_D for high-affinity sites was significantly increased in the frontal cortex and cerebellum in the presence of bicuculline (1 M) with no change in B_max. In contrast, the apparent affinity for low-affinity sites was not altered in the presence of bicuculline in these regions, whereas the B_max was significantly decreased in the cerebellum. Following acute (2 mg/kg, i.p.) or chronic (2 mg/kg, i.p. for 10 days) bicuculline treatment, [³H]SR 95531 binding was also investigated in various regions of brains. The acute bicuculline treatment did not affect the [³H]SR 95531 binding in any of the regions studied. In contrast, apparent affinity for [³H]SR 95531 was significantly decreased in low-affinity sites of all regions studied in rats treated chronically with bicuculline. The B_max values of high and low-affinity sites were significantly increased in the cerebellum with no change in the frontal cortex, striatum and substantia nigra. The present study demonstrates that chronic bicuculline treatment decreases apparent affinity of [³H]SR 95531 binding whereas the treatment increases apparent affinity of [³H]SR 95531 and [³H]muscimol binding in the cerebellum may be due to true up-regulation of GABA binding sites, involving increased de novo synthesis of receptor protein. These results also suggest that properties of cerebellar GABA_A receptors are different from those in other regions.Abbreviations used GABA -aminobutyric acid - FC frontal cortex - CB cerebellum - ST striatum - SN substantia nigra     

Chronic exposure of cells expressing recombinant GABAA receptors to benzodiazepine antagonist flumazenil enhances the maximum number of benzodiazepine binding sites

The aim of this study was to better understand the mechanisms that underlie adaptive changes in GABAA receptors following their prolonged exposure to drugs. Exposure (48 h) of human embryonic kidney (HEK) 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (1 or 5 microM) in the presence of GABA (1 microM) enhanced the maximum number (Bmax) of [3H]flunitrazepam binding sites without affecting their affinity (Kd). The flumazenil-induced enhancement in Bmax was not counteracted by diazepam (1 microM). GABA (1 nM-1 mM) enhanced [3H]flunitrazepam binding to membranes obtained from control and flumazenil-pretreated cells in a concentration-dependent manner. No significant differences were observed in either the potency (EC50) or efficacy (Emax) of GABA to potentiate [3H]flunitrazepam binding. However, in flumazenil pretreated cells the basal [3H]flunitrazepam and [3H]TBOB binding were markedly enhanced. GABA produced almost complete inhibition of [3H]TBOB binding to membranes obtained from control and flumazenil treated cells. The potencies of GABA to inhibit this binding, as shown by a lack of significant changes in the IC50 values, were not different between vehicle and drug treated cells. The results suggest that chronic exposure of HEK 293 cells stably expressing recombinant alpha1beta2gamma2S GABAA receptors to flumazenil (in the presence of GABA) up-regulates benzodiazepine and convulsant binding sites, but it does not affect the allosteric interactions between these sites and the GABA binding site. Further studies are needed to elucidate these phenomena.     

Solubilization and characterization of [3H]Imipramine and [3H]Paroxetine binding sites from calf striatum

The serotonin (5-HT) transporter from calf striatum cerebral membranes was solubilized with digitonin and characterized by gel exclusion chromatography. [³H]Imipramine and [³H]paroxetine were utilized as markers for labeling it.³H-imipramine labels a high- and a low-affinity site on striaturn membranes, whereas it binds to a single high-affinity site on the solubilized fraction. [³H]Paroxetine binds with the same affinity to a single site on both membranes and solubilized preparations. After gel exclusion chromatography of the solubilizate both [³H]imipramine and [³H]paroxetine bind on an identical fraction of 205 kDa molecular weight, with a similar maximum number of binding sites (Bmax). Our results suggest that both³H-imipramine and [³H]paroxetine bind to a common site on the 5-HT transporter.     

Resolution, configurational assignment, and enantiopharmacology of 2-amino-3-[3-hydroxy-5-(2-methyl-2H- tetrazol-5-yl)isoxazol-4-yl]propionic acid, a potent GluR3- and GluR4-preferring AMPA receptor agonist

We have previously shown that (RS)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol -4-yl] propionic acid (2-Me-Tet-AMPA) is a selective agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, markedly more potent than AMPA itself, whereas the isomeric compound 1-Me-Tet-AMPA is essentially inactive. We here report the enantiopharmacology of 2-Me-Tet-AMPA in radioligand binding and cortical wedge electrophysiological assay systems, and using cloned AMPA (GluR1-4) and kainic acid (KA) (GluR5, 6, and KA2) receptor subtypes expressed in Xenopus oocytes. 2-Me-Tet-AMPA was resolved using preparative chiral HPLC. Zwitterion (-)-2-Me-Tet-AMPA was assigned the (R)-configuration based on an X-ray crystallographic analysis supported by the elution order of (-)- and (+)-2-Me-Tet-AMPA using four different chiral HPLC columns and by circular dichroism spectra. None of the compounds tested showed detectable affinity for N-methyl-D-aspartic acid (NMDA) receptor sites, and (R)-2-Me-Tet-AMPA was essentially inactive in all of the test systems used. Whereas (S)-2-Me-Tet-AMPA showed low affinity (IC(50) = 11 microM) in the [(3)H]KA binding assay, it was significantly more potent (IC(50) = 0.009 microM) than AMPA (IC(50) = 0.039 microM) in the [(3)H]AMPA binding assay, and in agreement with these findings, (S)-2-Me-Tet-AMPA (EC(50) = 0.11 microM) was markedly more potent than AMPA (EC(50) = 3.5 microM) in the electrophysiological cortical wedge model. In contrast to AMPA, which showed comparable potencies (EC(50) = 1.3-3.5 microM) at receptors formed by the AMPA receptor subunits (GluR1-4) in Xenopus oocytes, more potent effects and a substantially higher degree of subunit selectivity were observed for (S)-2-Me-Tet-AMPA: GluR1o (EC(50) = 0.16 microM), GluR1o/GluR2i (EC(50) = 0.12 microM), GluR3o (EC(50) = 0.014 microM) and GluR4o (EC(50) = 0.009 microM). At the KA-preferring receptors GluR5 and GluR6/KA2, (S)-2-Me-Tet-AMPA showed much weaker agonist effects (EC(50) = 8.7 and 15.3 microM, respectively). It is concluded that (S)-2-Me-Tet-AMPA is a subunit-selective and highly potent AMPA receptor agonist and a potentially useful tool for studies of physiological AMPA receptor subtypes.     

高亲和力谷氨酸转运体结构和功能的研究进展

真核生物高亲和力谷氨酸转运体(excitatory amino acid transporters,EAATs)分为GLAST(EAAT1)、GLT-1(EAAT2)、EAAC1(EAAT3)、EAAT4和EAAT5等5个亚型.高亲和力谷氨酸转运体结构学的研究,揭示了谷氨酸转运体的跨膜拓扑结构、真核和原核生物EAATs结构的差异,以及在底物转运过程中的一些底物和协同转运离子的结合位点.其功能学的研究发现,EAATs在参与突触的传递,避免兴奋性氨基酸的毒性效应中发挥重要作用,同时也参与了对学习、记忆以及运动行为的调控.结合我们既往的工作,就近几年EAATs的结构和功能研究做一综述.     

"Specific" Binding of [3H]Imipramine to Protease-Sensitive and Protease-Resistant Sites

A number of 5-hydroxytryptamine (5-HT) uptake inhibitors have been shown to displace the binding of [3H]imipramine to rat cortical membranes in a complex manner with Hill slopes less than unity. Norzimeldine displaced the binding of [3H]imipramine in a biphasic manner with IC50 values for the two components of about 30 nM and 30 microM. This latter site alone was found in tissues that had been treated with a protease. Binding to both of these sites was displaced by 10 microM desipramine. The protease-sensitive [3H]imipramine binding sites were found to be saturable, high-affinity binding sites with a KD of 8 nM. The number of these sites varied between brain regions and was positively correlated with the regional distribution of [14C]5-HT but not [3H]noradrenaline uptake. This was not the case however for the protease-resistant but desipramine-displaceable binding sites. Since most previous [3H]imipramine binding studies have been performed with high concentrations of desipramine (10 microM) to define "specific binding," these data would suggest that either protease-sensitivity or displacability by 1 microM norzimeldine would give more reliable estimates of the specific binding.     

Quantitative autoradiography of Na+-dependent [3H]L-aspartate binding to L-glutamate transporters in rat brain: structure-activity studies using L-trans-pyrrolidine-2,4-dicarboxylate (L-t-PDC) and 2-(carboxycyclopropyl)-glycine (CCG)

Sodium-dependent binding of [3H]L-aspartate was studied in thaw-mounted horizontal sections of fresh-frozen (i.e. not fixed) rat brain. After the incubation with [3H]L-aspartate, the sections were exposed against a 3H-sensitive film and the resulting autoradiograms were evaluated by quantitative densitometry. Effects of several inhibitors were examined and their potency expressed as IC50 and nH. Together with previously published data, the present study supports the view that [3H]L-aspartate binding to fresh-frozen sections of rat brain represents interaction of the radioligand with the substrate-binding sites on glutamate transporters. The most potent inhibitors were (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and (2S,4R)-4-methylglutamate. In contrast, L-anti,endo-3,4-methanopyrrolidine dicarboxylate (L-a,e-MPDC) was about an order of magnitude less potent. Only subtle regional variations in the characteristics of inhibitors of [3H]L-aspartate binding were detected. It is not certain whether these differences reflect regional variations in the distribution of individual glutamate transporters or regional peculiarities in their pharmacological characteristics. In particular, (2S,4R)-4-methylglutamate, shown previously to differentiate between GLT-1 (principal glutamate transporter in the forebrain) and GLAST (expressed mainly in the cerebellum), did not strongly differentiate between the binding of [3H]L-aspartate in forebrain and cerebellum. Computer-assisted molecular modelling using selected glutamate analogues with restricted conformation (L-trans-pyrrolidine-2,4-dicarboxylate and four isomers of 2-(carboxycyclopropyl)-glycine: L- and D-CCG I, L-CCG III and L-CCG IV) identified at least one area of unfavourable steric interaction. We conclude that the quantitative autoradiographic studies using [3H]L-aspartate or other transporter-specific ligands, will be a useful tool to study the pharmacology of substrate binding sites on glutamate transporters in the mammalian brain in situ.