Inhibition by theanine of binding of [3H]AMPA, [3H]kainate,and [3H]MDL 105,519 to glutamate receptors

In an investigation of the mechanisms of the neuroprotective effects of theanine (gamma-glutamylethylamide) in brain ischemia, inhibition by theanine of the binding of [3H](RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, and [3H](E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1-H-indole-2-carboxylic acid (MDL 105,519) to glutamate receptors was studied in terms of its possible inhibiting effects on the three receptor subtypes (AMPA, kainate, and NMDA glycine), with rat cortical neurons. Theanine bound the three receptors, but its IC50 of theanine was 80- to 30,000-fold less than that of L-glutamic acid.     

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.     

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.     

Intracellular disposition of [3H]-cocaine, [3H]-norcocaine, [3H]-benzoylecgonine and [3H]-benzoylnorecgonine in the brain of rats

[³H]-cocaine, [³H]-norcocaine, [³H]-benzoylecgonine and [³H]-benzoylnorecgonine were administered i.c. in equi-potent pharmacologic doses and the intracellular disposition and metabolism of each drug determined. Norcocaine and cocaine rapidly entered and egressed from the brain so that 4.8–6.1% of the radioactivity present in brain at one minute was observed at 30 minutes. The highest levels of subcellular radioactivity were generally found in the microsomal plus supernatant, followed by the nuclear and shocked mitochondrial fractions. No apparent localization of the radioactivity occured in synaptic membranes. The brain/plasma (B/P) ratio curves for cocaine and norcocaine were similar; however, the norcocaine values were considerably higher at each time interval. Benzoylecgonine and benzoylnorecgonine had higher comparative B/P ratios than cocaine or norcocaine and persisted in brain for a longer period of time so that 0.6–2.1% of the radioactivity present in brain at 1 hour was detected at 24 hours. Cocaine and norcocaine were extensively metabolized to the benzoylmetabolites. Benzoylecgonine was metabolized to benzoylnorecgonine and benzoylnorecgonine was unmetabolized. The brain disposition data and B/P ratios agreed quite well with the overall pharmacologic action of cocaine and its metabolites.     

Analysis of [3',3'-d(2)]-nicotine and [3',3'-d(2)]-cotinine by capillary liquid chromatography-electrospray tandem mass spectrometry

A selective and sensitive LC/MS/MS assay was developed for the quantification of d(2)-nicotine and d(2)-cotinine in plasma of current and past smokers administered d(2)-nicotine. After solid phase extraction and liquid-liquid extraction, HPLC separation was achieved on a capillary hydrophilic interaction chromatography phase column. The analytes were monitored by tandem mass spectrometry with electrospray positive ionization. Linear calibration curves were generated for d(2)-nicotine (0.03-6.0 ng/ml plasma) and d(2)-cotinine (0.15-25 ng/ml plasma). The lower limits of quantitation were 0.15 ng/ml and 0.25 ng/ml for d(2)-nicotine and d(2)-cotinine, respectively. The coefficient of variation was 3.7% for d(2)-nicotine and 2.5% for d(2)-cotinine. The method was applied to two ongoing studies of d(2)-nicotine metabolism in prior and current smokers. Preliminary analysis of a subset of subjects from these studies detected a significantly lower rate of nicotine conversion to cotinine by past smokers compared to current smokers.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Changes of [3H]MK-801, [3H]muscimol and [3H]flunitrazepam Binding in Rat Brain by the Prolonged Ventricular Infusion of Transformed Ginsenosides

Ameliorating effects of ginseng were observed on neuronal cell death associated with ischemia or glutamate toxicity. Ginseng saponins are transformed by intestinal microflora and the transformants would be absorbed from intestine. In the present study, we have investigated the effects of transformed ginsenoside Rg3, Rh2 and compound K on the modulation of NMDA receptor and GABA_A receptor binding in rat brain. The NMDA receptor binding was analyzed by quantitative autoradiography using [³H]MK-801 binding, and GABA_A receptor bindings were analyzed by using [³H]muscimol and [³H]flunitrazepam binding in rat brain slices. Ginsenoside Rg3, Rh2 and compound K were infused (10 g/10 l/h) into rat brain lateral ventricle for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]MK-801 binding were highly decreased in almost all regions of frontal cortex and hippocampus by ginsenoside Rh2 and compound K. The levels of [³H]muscimol binding were elevated in part of frontal cortex and granule layer of cerebellum by the treatment of ginsenoside Rh2 and compound K. However, the [³H]flunitrazepam binding was not modulated by any tested ginsenosides. Ginsenoside Rh2 and compound K induced the downregulation of the [³H]MK-801 binding as well as upregulation of the and [³H]muscimol binding in a region-specific manner after prolonged infusion into lateral ventricle. However, ginsenoside Rg3 did not show the significant changes of ligand bindings. In addition, ginsenoside Rh2 decreased the expression of nNOS in the hippocampus although Rg3 decreased the expression in the cortex. These results suggest that biotransformed ginsenoside Rh2 and compound K could play an important role in the biological activities in the central nervous systems and neurodegenerative disease.     

Syntheses of 24R,25-dihydroxy-[6,19,19-3H]vitamin D3 and 24R,25-dihydroxy-[6,19,19-2H]vitamin D3

24R,25-Dihydroxy-[6,19,19-3H]vitamin D3 with a specific activity of 54 Ci/mmol and 24R,25-dihydroxy-[6,19,19-2H]vitamin D3 with 2.6 deuterium atoms/mol were synthesized in four steps starting from 24R,25-Dihydroxyvitamin D3 via its sulfur dioxide adduct.     

The synthesis of high-specific-activity UDP-[6-3H]galactose, UDP-N-[6-3H]acetylgalactosamine, and their corresponding monosaccharides.

Tritiated uridine-5'-diphosphogalactose (UDP-[3H]Gal) has been widely used to study oligosaccharide biosynthesis and structure. It can be synthesized either chemically or enzymatically using galactose oxidase to oxidize the hydroxyl moiety at C-6 to an aldehyde (6-aldo-UDP-Gal), which is then reduced back to the alcohol with tritiated sodium borohydride. Although the enzymatic approach is simple and efficient, there are several problems associated with it. First, incomplete oxidation to the aldehyde reduces the final specific activity. Second, if the galactose oxidase is not removed from the 6-aldo-UDP-Gal prior to reduction, the resulting UDP-[6-3H]Gal can be reoxidized to 6-aldo-UDP-[6-3H]Gal. We present evidence for the occurrence of this compound in one commercially obtained preparation of UDP-[6-3H]Gal. Finally, if an excess of 6-aldo-UDP-Gal is used for good yield, it is necessary to quench the reduction with nonradioactive borohydride, again reducing the final specific activity. We have devised a rapid, inexpensive, and efficient synthesis of UDP-[6-3H]Gal that circumvents all of these problems. Galactose oxidase is used to produce 6-aldo-UDP-Gal and the completeness of this reaction is confirmed on polyethyleneimine (PEI) cellulose TLC plates. The 6-aldo-UDP-Gal is purified on silica gel 60 TLC plates. This purified compound is then reduced with tritiated sodium borohydride, with the aldehyde present in excess. Unreacted 6-aldo-UDP-Gal is then purified away from the product UDP-[6-3H]Gal by chromatography on PEI cellulose. Radiochemically pure UDP-[6-3H]Gal with a specific activity of 10 Ci/mmol was obtained using the above scheme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative binding properties of linear and cyclic delta-selective enkephalin analogues: [3H]-[D-Thr2, Leu5] enkephalyl-Thr6 and [3H]-[D-Pen2, D-Pen5] enkephalin

The range of delta-selectivity of linear and cyclic analogues of enkephalin in rat brain was found to be: [D-Pen2, L-Pen5] enkephalin (DPLPE) greater than [D-Pen2, D-Pen5] enkephalin (DPDPE) greater than [D-Thr2, Leu5] enkephalyl-Thr6 (DTLET) greater than [D-Ser2, Leu5] enkephalyl-Thr6 (DSLET). Saturation experiments performed with [3H]DPDPE and [3H]DTLET in NG108-15 cells and rat brain showed similar binding capacities for both the ligands, but the delta-affinity of [3H]DTLET (KD approximately 1.2 nM) was much better than that of [3H]DPDPE (KD approximately 7.2 nM). The rather low delta-affinity of DPDPE induced high experimental errors cancelling the benefit of its better delta-selectivity. Binding experiments in rat or guinea-pig brains showed, in both cases, the better delta-selectivity of [3H]DTLET compared to [3H]DSLET. The former peptide remains at this time the most appropriate radioactive probe for binding studies of delta-receptor.     

DNA crosslinks,single-strand breaks and effects on bacteriophage T4 survival from tritium decay of [2-3H]adenine, [8-3H]adenine and [8-3H]guanine

Escherichia coli and bacteriophage T4 DNA containing [2-³H]adenine accumulated crosslinks between the complementary strands. For T4 DNA stored in frozen solution there were 0.41 to 0.54 crosslinks formed per tritium decay. The crosslinks were demonstrated both by an increased DNA sedimentation rate in alkaline sucrose gradients and by an increasing amount of DNA that renatured quickly after denaturation by heat or alkali. Single-strand breaks were also formed with an efficiency of 0.08 to 0.50 breaks per tritium decay. DNA containing both [8-³H]adenine and [8-³H]guanine showed no crosslinking but did undergo single-strand breaks at a rate of 0.08 per tritium decay. T4 bacteriophage containing [2-³H]adenine lost plaque-forming ability when stored at 4 °C, with 0.34 lethal hits per tritium decay, whereas the same phage labeled with a mixture of [8-³H]adenine and [8-³H]guanine sustained only 0.12 lethal hits per tritium decay. The loss of plaque-forming ability in the latter case is probably due to a radiation effect from the emitted beta particle; the high lethal efficiency for tritium decay at 2-adenine is probably caused either by crosslinks between complementary strands or from some undetected lesion produced in the DNA.     

Basipetally polarised transport of [3H]gibberellin A1 and [14C]gibberellin A3, and acropetal polarity of [14C]indole-3-acetic acid transport,in stelar tissues of Phaseolus coccineus roots

Summary Movement of both [³H]GA₁ and [¹⁴C]GA₃ through root segments from P. coccineus seedlings was basipetally polarised. The basipetal/acropetal ratio of radioactivity from [³H]GA₁ in agar receiver blocks was 9.2 for apical, elongating segments, and 4.0 for more basal, non-elongating segments. Polarity of gibberellin transport was restricted to the stele, and absent from cortical tissues. Transport of [¹⁴C]IAA through root segments to agar receivers was preferentially acropetal, particularly so in the stele. Despite the existence of basipetal polarity of gibberellin transport in the root, [³H]GA₁ injected into cotyledons moved into and acropetally along the seedling root.     

[3H]Tyramine binding: A comparison with neuronal [3H]-dopamine uptake and [3H]mazindol binding processes

[³H]Spiperone ([³H]SPI) binding sites in rat or bovine striata have been solubilized using CHAPS or digitonin detergents. Solubilized sites retained the binding characteristics of those in native membrane preparations. The same solubilized material, however, did not bind [³H]tyramine ([³H]PTA), thus indicating that [³H]PTA binding sites and DA receptors are different chemico-physical entities. In membrane preparations or crude synaptosomes obtained from the c.striatum of neonatally-rendered hypothyroid rats, when central DA-pathways are impaired, both [³H]PTA binding and [³H]DA uptake processes were markedly decreased, with no effect on [³H]mazindol ([³H]MAZ) binding, compared to euthyroids. Reserpine, a well-known inhibitor of DA-uptake into a variety of secretory vesicles, and a potent in vivo andin vitro inhibitor of [³H]PTA binding, did not affect the [³H]MAZ binding process. This further supported the suggestion that while [³H]PTA binding sites are almost totally associated with the vesicular transporter for DA, [³H]MAZ does label a site involved in the DA-translocation across the neuronal membrane. The latter process seems to be rather insensitive to thyroid hypofunction, when however the intracellular storage of DA might be consistently impaired. In conclusion, PTA might be well exploited as a marker of the DA vesicular transporter through its molecular characterization, whenever possible.Special issue dedicated to Dr. Paola S. Timiras     

Conjugated forms of [3H] alpha, 25-dihydroxyvitamin D3 in rat bile

When small doses of [3H]D3, [3H]25-OHD3 and [3H]alpha, 25-diOHD3 were administered intravenously to rats 6.3 +/- 1.1% (means +/- SEM, n = 4), 9.7 +/- 0.9% (n = 6) and 12.8 +/- 2.6% (n = 8), respectively, of the administered radioactivity was excreted in bile. The radioactive biliary conjugated metabolites were analysed by ion exchange chromatography: in the case of all 3 substrates about 30% of metabolites were found to be cationic on the basis of their being retained on sulphopropyl-Sephadex G-25 (H+-form) when applied in 70% methanol. The balance of the metabolites were neutral and anionic and were analysed on TEAP-Lipidex: in the case of 1 alpha, 25-diOHD3 the following metabolite classes were detected (on the basis of co-elution with authentic standards) (in order of quantitative importance): taurine conjugates, neutral metabolites, monosulphates, glucuronides, carboxylic acids, glycine conjugates and disulphates. Alkaline hydrolysis of the taurine and glycine conjugates yielded products 60% of which now chromatographed as carboxylic acids. Hydrolysis of the glucuronide and monosulphate fractions indicated significant levels of mixed conjugation yielding some products which now chromatographed as glycine and taurine conjugates, respectively. The nature of the cationic conjugates was not elucidated but they had the following properties: they could be hydrolysed by alkali to yield non-cationic radioactive metabolites (these released metabolites were heterogeneous as judged by TEAP-lipidex chromatography); they were partially hydrolysed to non-cationic forms by beta-glucuronidase; and on reverse-phase HPLC they had an elution profile that was significantly different to that of histidyl-, ornithyl- or lysyl-calcitroic acid.