Characterization of a specific L-[3H]glutamic acid binding site on cilia isolated fromParamecium tetraurelia

Summary Cilia isolated fromParamecium tetraurelia possess a specific, high affinity L-[³H]glutamic acid binding site, defined by an ED₅₀ of 3.0×10^–8 M. The structural specificity of this site was probed by testing the competition between L-glutamate and various analogues for binding to cilia. The binding site is stereo-specific for L-glutamic acid, and requires the presence of all three ionizable groups on the glutamate molecule for optimal ligand: receptor interaction.Specific binding of L-[³H]glutamic acid to cilia is rapid in onset but transient, reaching peak values within 6 min, and then declining thereafter. This transience may represent a form of sensory adaptation during prolonged exposure to the ligand.     

Synthesis of an acromelic acid A analog-based 11C-labeled PET tracer for exploration of the site of action of acromelic acid A in allodynia induction

A novel ¹¹C-labeled PET (positron emission tomography) tracer, which was designed based on the (phenylthio)pyrrolidine derivative that can competitively block the acromelic acid A-induced allodynia, was synthesized. A protocol in which methylation by palladium-mediated coupling of the boronate derivative with [¹¹C]CH₃I and deprotection of the protected amino acid moiety are successively performed in one-pot within 5 min was established for the synthesis of the tracer. The tracer is potentially useful as a tool to investigate the mode of action of acromelic acid A in the induction of allodynia.     

Human, Bovine, and Rabbit Retinal Glutamate-Induced [3H]D-Aspartate Release: Role in Excitotoxicity

The pharmacological basis of glutamate-induced [³H]D-aspartate release was investigated in isolated human, bovine and rabbit retinas. Isolated mammalian retinas were preloaded with [³H]D-aspartate and then prepared for studies of neurotransmitter release using the superfusion method. Release of [³H]D-aspartate was elicited by K⁺ (50 mM) or by L-glutamate. In bovine retinas, L-glutamate, but not D-glutamate induced an overflow of [³H]D-aspartate that was partially inhibited by low external calcium, -conotoxin (10 nM) or nitrendipine (1 M). Metabotropic glutamate receptor (GLUR) agonists also evoked [³H]D-aspartate release in both bovine and human retinas whereas polyamines only enhanced the excitatory effects of L-glutamate on [³H]D-aspartate release. Antagonists of GLURs and the polyamine site inhibited L-glutamate evoked [³H]D-aspartate overflow with the following rank order of potency: MCPG >ifenprodil > AP-5 > arcaine> MK-801. In conclusion, L-glutamate-induces a stereoselective, calcium-dependent release of [³H]D-aspartate from isolated mammalian retinas that can be mimicked by GLUR agonists (and blocked by both receptor and polyamine site antagonists).     

Amino acid uptake by neurones and glial cells from embryonic cockroach brain growing in vitro

Insect neuronal cultures and glial-enriched cultures derived from the brains of embryonic cockroaches have been used to investigate the uptake of putative amino acid neurotransmitters. Some neurones and all of the glia in these cultures possess uptake systems for [³H]l-glutamate and [³H]GABA. The neuronal and glial uptake of l-glutamate is reduced by d-aspartate. Neuronal accumulation of [³H]GABA is reduced by nipecotic acid but is not inhibited by β-alanine and DABA, whereas DABA is the most potent inhibitor of GABA accumulation by glia. The cockroach glial cells did not accumulate taurine, glycine, d-aspartate or leucine but there was some neuronal uptake of both taurine and glycine although this was insensitive to sodium ions.     

Inositol trisphosphate mediates the action of hypertrehalosemic hormone on fat body of the American cockroach, Periplaneta americana

The rate of synthesis of inositol trisphosphate (InsP₃) in trophocytes derived from disaggregated cockroach (Periplaneta americana) fat body increases following treatment of the cells with hypertrehalosemic hormone I or II (HTH-I, -II) in vitro. Trophocytes preloaded with [³H]inositol display a significant increase in InsP₃ synthesis as early as 15 s after addition of the hormone. When the trophocytes are pre-incubated with LiCl and subsequently incubated with HTH the [³H] content of the InsP₃ fraction is greater than that found with HTH alone. This is taken as evidence that inositol monophosphate phosphatase is part of the mechanism for clearing InsP₃ from the cytosol. In contrast to HTH, octopamine, which is also capable of exerting a hypertrehalosemic effect in the cockroach, does not increase the synthesis of InsP₃. 1-Octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH₃), a potent and selective inhibitor of phosphatidylinositol phospholipase C, blocks the activation of phosphorylase by HTH-I as well as the hypertrehalosemic effect induced by the hormone.     

Support for Radiolabeling of a Glycine Recognition Domain on the N-Methyl-d-Aspartate Receptor Ionophore Complex by 5,7-[3H]Dichlorokynurenate in Rat Brain

Abstract: Pretreatment with Triton X-100 more than doubled the binding of radiolabeled 5,7-dichlorokynurenic acid (DCKA), a proposed antagonist at a glycine (Gly) recognition domain on the N-methyl-d -aspartate (NMDA) receptor ionophore complex, in rat brain synaptic membranes. The binding exhibited an inverse temperature dependency, reversibility, and saturability, the binding sites consisting of a single component with a high affinity (27.5 nM) and a relatively low density (2.87 pmol/mg of protein). The binding of both [³H]DCKA and [³H]Gly was similarly displaced by numerous putative agonists and antagonists at the Gly domain in a concentration-dependent manner at a concentration range of 100 nM to 0.1 mM. Among the 24 putative ligands tested, DCKA was the second most potent displacer of the binding of both radioligands with no intrinsic affinity for the binding of [³H]kainic acid and α-amino-3-hydroxy-5-[³H]methylisoxazole-4-propionic acid (AMPA) to the non-NMDA receptors. In contrast, the other proposed potent Gly antagonist, 5,7-dinitroquinoxaline-2,3-dione, was active in displacing the binding of [³H]glutamic ([³H]Glu) and D,L-(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acids to the NMDA recognition domain with a relatively high affinity for the non-NMDA receptors. In addition, the proposed antagonist at the AMPA-sensitive receptor, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline, not only displaced weakly the binding of both [³H]- Gly and [³H]DCKA, but also inhibited the binding of (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the NMDA-sensitive receptor in the presence of added Glu alone in a manner sensitive to antagonism by further added Gly. Clear correlations were seen between potencies of the displacers to displace [³H]DCKA binding and [³H]Gly binding, in addition to between the potencies to displace [³H]-DCKA or [³H]Gly binding and to potentiate or inhibit [³H]MK-801 binding. All quinoxalines tested were invariably more potent displacers of [³H]DCKA binding than [³H]Gly binding, whereas kynurenines were similarly effective in displacing the binding of both [³H]Gly and [³H]-DCKA. These results undoubtedly give support to the proposal that [³H]DCKA is one useful radioligand available in terms of its high selectivity and affinity for the Gly domain in the brain. Possible multiplicity of the Gly domain is suggested by the differential pharmacological profiles between the binding of [³H]Gly and [³H]DCKA.     

Characterisation of [3H]MK-801 Binding and its Cooperative Modulation by PIG Brain Membranes

Abstract

Cooperative modulation of [³H]MK-801 binding to extensively washed pig cortical brain membranes in the presence of various concentrations of L-glutamate, glycine, spermine, CPP and DCKA was evaluated in association experiments. In saturation experiments [³H]MK-801 labelled a homogeneous population of binding sites with a K_d-value of 1.26 ± 0.18 nmol 1^?1 and a B_max-value of 2130 ± 200 fmol/mg protein. The pharmacological profile of this site was further evaluated in competition experiments with known NMDA receptor channel blockers. In nonequilibrium binding experiments EC₅₀-values of reference compounds acting at the L-glutamate, at the glycine, and at the polyamine site, were determined by increasing or decreasing [³H]MK-801 binding. Ifenprodil reduced [³H]MK-801 binding in a biphasic manner. All the data obtained are in agreement with results from [³H]MK-801 binding to rodent as well as human brain membranes. This study therefore strongly suggests, that pig cortical membranes are a suitable alternative to rodent brain membranes, and an acceptable substitute for human brain membranes in [³H]MK-801 binding experiments.     

Further Evidence for Multiple Forms of an N-Methyl-d-Aspartate Recognition Domain in Rat Brain Using Membrane Binding Techniques

Abstract— Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsul-fonic acid, invariably resulted in marked inhibition of the binding of dl -(E)-2-amino-4-[³H]propyl-5-phosphono-3-pentenoic acid ([³H]CGP 39653), a competitive antagonist at an N-methyl-d -aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X-100, but did not significantly affect the binding of L-[³H]-glutamic acid ([³H]Glu), an endogenous agonist. The pre-treatment was effective in reducing the binding of [³H]-CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl-reactive agents also enhanced the potencies of NMDA agonists to displace [³H]-CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [³H]Glu binding. The binding of both [³H]CGP 39653 and [³H]Glu was similarly sensitive to pretreatment with four different proteases in Tritontreated membranes, whereas pretreatment with phospho-lipase A₂ or C markedly inhibited [³H]CGP 39653 binding without altering [³H]Glu binding. Moreover, both phospho-lipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [³H]CGP 39653 and [³H]Glu, but also caused diminution of those of NMDA antagonists. These results suggest that both sulfhydryl-reactive agents and phospholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [³H]CGP 39653, with concomitant facilitation of that in an agonist-preferring form by [³H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.     

Binding sites for [3H]GABA, [3H]flunitrazepam and [35S]TBPS in insect CNS

The CNS of the cockroach Periplaneta americana contains saturable, specific binding sites for [³H]GABA, [³H]flunitrazepam and [³⁵S]TBPS. The [³H]GABA binding site exhibits a pharmacological profile distinct from that reported for mammalian GABA_A and GABA_B receptors. The most potent inhibitors of [³H]GABA binding were GABA and muscimol, whereas isoguvacine, thiomuscimol and 3-aminopropane sulphonic acid were less effective. Bicuculline methiodide and baclofen were ineffective. Binding of [³⁵S]TBPS was partially inhibited by 1.0 × 10⁻⁶ M GABA, whilst binding of [³H]flunitrazepam was enhanced by 1.0 × 10⁻⁷ M GABA. The pharmacological profile of the [³H]flunitrazepam binding site showed some similarities with the peripheral benzodiazepine binding sites of vertebrates, with Ro-5-4864 being a far more effective inhibitor of binding than clonazepam. Thus a class of GABA receptors with pharmacological properties distinct from mammalian GABA receptor subtypes is present in insect CNS.     

Metabolism of juvenile hormone in cultures of ovaries and fat body in the cockroachperiplaneta Americana

Summary The time course of juvenile hormone (JH) metabolism is examined in cultures ofPeriplaneta americana fat body and ovaries in medium containingManduca sexta carrier protein or cockroach hemolymph. In the absence ofM. sexta carrier protein or cockroach hemolymph, both tissues extensively catabolize exogenous [³H]JH in the medium. Addition of the carrier protein or hemolymph to the culture system prevents the hydrolysis of the hormone in the medium. Within the tissues JH is degraded whether or not carrier protein or hemolymph is present which suggests that the protective role of these molecules is exclusively extracellular. Incubation of [³H]JH with medium preconditioned with tissue results in destruction of the hormone. This suggests that the fat body secretes esterases into the medium. In contrast, the ovarioles hydrolyze the hormone by means of cell-associated enzyme. The relationship of these phenomena to insect development is discussed. This work supported by NSF Grant PCM 76-02229 and University of Kansas Biomedical Sciences Grant RR-07037.     

Transport of monoamine and amino acid neurotransmitters by primary astroglial cultures

The transport of [³H]l-glutamate, [³H]l-aspartate, [³H]-aminobutyric acid ([³H]GABA), [³H]dopamine, [³H]norepinephrine and [³H]5-hydroxytryptamine (³H-5-HT) was measured in primary astroglial cultures from newborn rat cerebral hemispheres. There was a high-affinity uptake with aK _m of 69.0 M for L-glutamate, 12.3 M forl-aspartate and 3.1 M for GABA. The uptake showed properties of high capacity with aV _max of 17.0 nmol·mg prot^–1·min^–1 forl-glutamate, 1.1 nmol·mg prot^–1·min^–1 forl-aspartate and 0.04 nmol·mg prot^–1·min^–1 for GABA. No high-affinity high capacity transport system was found for the monoamines studies. Autoradiographic examination demonstrated a heavy deposit of grains suggesting a prominent accumulation of [³H]l-glutamate and [³H]l-aspartate in the astroglial-like cells of the cultures, while the [³H]GABA accumulation was less intense. On the other hand, there was only a weak accumulation of grains after incubating the cultures with [³H]dopamine, [³H]norepinephrine or [³H]5-HT. Thus, astroglial cells in culture accumulate amino acid neurotransmitters and monoamines in different ways with a high-affinity high-capacity uptake of glutamate, aspartate and GABA and a diffusion-uptake of dopamine, norepinephrine and 5-HT.     

Autoradiographic localization of [3H]-GABA accumulation in the sixth abdominal ganglion of the cockroach, Periplaneta americana L.

Localization of [³H]-GABA accumulation in the sixth abdominal ganglion of the cockroach was achieved using autoradiography.Light and electron microscope autoradiographic results provide direct evidence for glial accumulation of [³H]-GABA via a Na⁺-dependent carrier system. The functional role of the glial uptake of GABA is discussed.     

Characterization of the Influence of Unsaturated Free Fatty Acids on Brain GABA/Benzodiazepine Receptor Binding In Vitro

Abstract: We have investigated the effect of unsaturated free fatty acids (FFAs) on the brain GABA/benzodiazepine receptor chloride channel complex from mammalian, avian, amphibian, and fish species in vitro. Unsaturated FFAs with a carbon chain length between 16 and 22 carbon atoms enhanced [³H]diazepam binding in rat brain membrane preparations, whereas the saturated analogues had no effect. The enhancement of [³H]diazepam binding by oleic acid was independent of the incubation temperature (0-30°C) of the binding assay and not additive to the enhancement by high concentrations of C1. In rat brain preparations, the stimulation of [³H]diazepam binding by oleic acid (10^?4M) was independent of the ontogenetic development. Phylogenetically, large differences were found in the effect of unsaturated FFAs on [³H]diazepam and [³H]muscimol binding: In mammals and amphibians, unsaturated FFAs enhanced both [³H]-muscimol and [³H]diazepam binding to 150-250% of control binding. In 17 fish species studied, oleic acid (10^?4M) stimulation of [³H]diazepam binding was weak (11 species), absent (four species), or reversed to inhibition (two species), whereas stimulation of [³H]muscimol binding was of the same magnitude as in mammals and amphibians. In 10 bird species studied, only weak enhancement of [³H]muscimol binding (110–130% of control) by oleic acid (10^?4M) was found, whereas [³H]diazepam binding enhancement was similar to values in mammal species. Radiation inactivation of the receptor complex in situ from frozen rat cortex showed that the functional target size for oleic acid to stimulate [³H]flunitrazepam binding has a molecular mass of ～200,000 daltons. Our data show that unsaturated FFAs have distinct effects on membranebound GABA/benzodiazepine receptors in vitro.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Characterization of phenylalkylamine binding sites in insect (Periplaneta americana) nervous system and skeletal muscle membranes

This study has identified specific, stereoselective phenylalkylamine (PAA, (±)- [³H]verapamil) binding sites of low-affinity and high-density in cockroach (Periplaneta americana) nervous system and skeletal muscle membranes. Scatchard transformation of equilibrium binding data revealed a single population of binding sites in both tissues with dissociation constants (K_d) of 273 nM and 377 nM and binding capacities (B_max) of 23 pmol·mg protein^?1 and 37pmol·mg protein^?1 for cockroach nervous tissue and skeletal muscle membranes, respectively. The PAA binding site in cockroach nervous tissue membranes was found to be dihydropyridine (DHP)-insensitive, whereas the corresponding site in cockroach skeletal muscle membranes was DHP-sensitive. This property of a DHP-sensitive PAA receptor distinguishes the binding sites identified in cockroach skeletal muscle from those in cockroach nervous tissue and indicates that pharmacologically distinct putative Ca²⁺ channel subtypes are present in insect nerve and muscle. © 1993 Wiley-Liss, Inc.     

A Possible Role of Glutathione as an Endogenous Agonist at the N-Methyl-d-Aspartate Recognition Domain in Rat Brain

Abstract: Glutathione, both reduced (GSH) and oxidized (GSSG), was effective in displacing binding of l -[³H]-glutamic acid (l -[³H]Glu) and dl -(E)-2-[³H]amino-4-propyl-5-phosphono-3-pentenoic acid ([³H]CGP-39653) in rat brain synaptic membranes, with less potent displacement of binding of dl -α-amino-3-hydroxy-5-[³H]-methylisoxazole-4-propionic and [³H]kainic acids. Liquid chromatographic analysis revealed that both GSH and GSSG were contaminated with l -Glu by <1%. Both GSH and GSSG potentiated (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) binding in a manner similar to that found with l -Glu. Pre-treatment with glutamate dehydrogenase (GDH) induced a marked rightward shift of the concentration-response curve for l -Glu in the presence of NAD without affecting that in its absence, whereas GDH was ineffective in affecting the potentiation by both GSH and GSSG even in the presence of NAD. In the presence of GSH at a maximally effective concentration, both glycine (Gly) and spermidine potentiated [³H]MK-801 binding to a somewhat smaller extent than that found in the presence of l -Glu at a maximally effective concentration. The potentiation of [³H]MK-801 binding by GSH was invariably attenuated by addition of CGP-39653, d -2-amino-5-phosphonovaleric acid (d -AP5), and 5,7-dichlorokynurenic acid (DCKA), whereas GSH was effective in diminishing potencies of CGP-39653, d -AP5, DCKA, and 6,7-dichloroquinoxaline-2,3-dione to inhibit [³H]MK-801 binding when determined in the presence of both l -Glu and Gly. These results suggest that glutathione may be an endogenous agonist selective for the N-methyl-d -aspartate (NMDA) recognition domain on the NMDA receptor ionophore complex.     

Biosynthesis of indole-3-acetic acid in protoplasts,chloroplasts and a cytoplasmic fraction from barley (Hordeum vulgare L.)

Protoplast preparations from barley (Hordeum vulgare L.) enzymatically converted [5-³H]tryptophan to [³H]indole-3-acetic acid (IAA). Both a chloroplast and a crude cytoplasmic fraction, isolated from protoplasts that had previously been fed [5-³H]tryptophan, contained [³H]IAA. Chloroplast and cytoplasmic preparations, isolated from protoplasts and thereafter incubated with [5-³H]tryptophan, also synthesized [³H]IAA, although, in both instances the pool size was less than 50% of that detected in the in-vivo feeds. There were no significant differences in the amounts of [³H]IAA that accumulated in protoplast and chloroplast preparations incubated in light and darkness.Abbreviations HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - RC radiocounting     

Developmental changes in high-affinity uptake of GABA by cultured neurons

High-affinity uptake of [³H]-aminobutyric acid (GABA) was studied in cultures of neonatal rat cortical neurons grown on pre-formed monolayers of non-neuronal (glial) cells. Both the maximum rate (V _max) and, to a smaller extent, theK _m of [³H]GABA uptake increased with time. In addition, in parallel with these changes, 2,4-diaminobutyric acid and cis-3-aminocyclohexane-1-carboxylic acid (ACHC), compounds which are considered typical substrate/inhibitors of GABA uptake in neurons, became progressively stronger inhibitors of [³H]GABA uptake. Consequently, the present results may mean that the studies using uptake, of [³H]GABA, [³H]ACHC, or [³H]DABA as a specific marker for GABAergic neurons differentiating during the ontogenetic development of the central nervous system may have to be interpreted with caution.     

[N-Methyl-3H]Scopolamine binding sites in the central nervous system of the cockroach Periplaneta americana

The binding of [N-methyl-³H]scopolamine to a cockroach nerve cord preparation has been investigated. Specific [N-methyl-³H]scopolamine binding was found to be saturable and of high affinity (K_d = 13.9 nM). Muscarinic ligands were found to displace [N-methyl-³H]scopolamine binding more effectively than nicotinic ligands. The distribution of these [N-methyl-³H]scopolamine binding sites was examined in the metathoracic ganglion at the light microscope level by autoradiographical techniques. Specific binding was found to be localized to distinct regions of the neuropile. This pattern showed certain similarities to that seen when the ganglion was stained for acetylcholinesterase, suggesting a functional role for these insect muscarinic acetylcholine receptors.     

Binding of [3H]phencyclidine to membranes from housefly thoracic muscles

The binding of [³H]phencyclidine ([³H]PCP) to a preparation of housefly thoracic muscle membranes was studied. Specific [³H]PCP binding saturated with both time and at concentrations of the radiolabeled ligand greater than 20 nM. One binding site with a K_D of 10.7 nM and a B_max of 3.9 pmol/mg protein was observed. Specific [³H]PCP binding was also readily reversible with a half-time of dissociation (t_1/2) of 13.8 min and varied proportionately with tissue concentration. Of the drugs tested, specific [³H]PCP binding was inhibited by PCP analogs, antipsychotics, antidepressants, Ca²⁺ channel antagonists, and K⁺ channel blockers. [³H]PCP binding was unaffected by addition of carbamylcholine or L-glutamate in absence or presence of ATP, GTP, cAMP, or cGMP. Though the identity of the [³H]PCP binding protein in housefly muscle membranes is still unclear, it is more likely to be an ionic channel such as K⁺ or Ca²⁺ channels than a neurotransmitter receptor.