共查询到20条相似文献,搜索用时 15 毫秒
1.
Abstract: The multisubunit γ-aminobutyric acid type A (GABAA ) receptor is heterogeneous in molecular and pharmacological aspects. We used quantitative autoradiographic techniques to generate detailed pharmacological profiles for the binding of the GABAA -receptor ionophore ligand tert -[35 S]butylbicyclophosphorothionate ([35 S]TBPS) and its modulation by GABA and the GABAA antagonists bicuculline and 2'-(3'-carboxy-2',3'-propyl)-3-amino-6- p -methoxyphenylpyrazinium bromide (SR 95531). Regional differences in the actions of bicuculline and SR 95531 were correlated with the expression of 13 GABAA subunits in brain as reported previously. In some brain regions SR 95531 reduced [35 S]TBPS binding much more than bicuculline, as illustrated by high ratios of bicuculline- to SR 95531-modulated [35 S]TBPS binding. This ratio correlated positively with α2-subunit mRNA levels. Binding that was equally affected by SR 95531 and bicuculline occurred prominently in regions with abundant α1 mRNA expression. The present findings thus reveal a novel pharmacological heterogeneity based on differences between α1 and α2 subunit-containing GABAA receptors. The data aid in developing GABAA -receptor subtype-specific antagonists and in establishing receptor domains critical for the actions of GABAA antagonists. 相似文献
2.
The sulfating system in sea urchin embryos was examined, using the labeled precursor inorganic [35 S]sulfate in vivo and [35 S]3'-phosphoadenosine 5'-phosphosulfate ([35 S]PAPS) in a cell-free system. In vivo incorporation of [35 pS]sulfate into the trichloroacetic acid (TCA)-insolubte fraction increased gradually during sea urchin development, whereas radioactivity of [35 S]sulfate contained in the TCA-soluble fraction showed a conspicuous peak at the late gastrula stage.
In a cell-free system, the particulate fraction showed marked incorporation of [35 pS]JPAPS. This sulfating activity was highest at pH 6.4 to 7.2 and at 27°C, and it was strongly inhibited by Hg 2+ and p-chloromercuribenzoic acid.
The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage. 相似文献
In a cell-free system, the particulate fraction showed marked incorporation of [
The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage. 相似文献
3.
Christiane Gulat-Marnay rée Lafitte Froylan Vargas Jean-Charles Schwartz 《Journal of neurochemistry》1987,49(5):1443-1448
Abstract: The accumulation and utilization of [35 S]3'-phos-phoadenosine 5'-phosphosulfate (PAPS) were studied in slices from rat cerebral cortex incubated in the presence of inorganic [35 S]sulfate. [35 S]PAPS levels were directly evaluated after either isolation by ion-exchange chromatography or quantitative enzymatic transfer of its active [35 S]sulfate group to an acceptor phenol under the action of added phenolsulfotransferase activity. [35 S]PAPS formation was also indirectly followed by incubating slices in the presence of β-naphthol and measuring the levels of [35 S]β-naphthyl sulfate ([35 S]β-NS). Whereas [35 S]PAPS levels rapidly reached a plateau, [35 S]β-NS formation proceeded linearly with time for at least 1h, an observation indicating that the nucleotide was continuously synthesized and utilized for endogenous sulfation reactions. [35 S]PAPS formation in ices was completely and rather potently blocked by 2,6-dichloro-4-nitrophenol (IC50 = .10 μM), an inhibitor of the PAPS-synthesizing enzyme system in a cytosolic preparation. [35 S]PAPS accumulation and [35 S]β-NS'formation were strongly reduced by depolarizing agents such as potassium or veratridine. At millimolar concentrations, various excitatory amino acids (glutamate, aspartate, cysteate, quisqualate, and homocysteate) also elicited similar effects, whereas kainate and N -methyl-D-aspartate were inactive. This suggests that PAPS synthesis is turned off when cerebral cells are strongly depolarized. 相似文献
4.
Laura J. Sim Qixu Liu Steven R. Childers Dana E. Selley 《Journal of neurochemistry》1998,70(4):1567-1576
Abstract: Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ-opioid receptors. In the present study, the effect of endomorphin-1 on μ receptor-coupled G proteins was compared with that of the μ agonist DAMGO by using agonist-stimulated [35 S]GTPγS binding in rat brain. [35 S]GTPγS autoradiography revealed a similar localization of endomorphin-1 and DAMGO-stimulated [35 S]GTPγS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [35 S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [35 S]GTPγS binding. Differences in maximal stimulation of [35 S]GTPγS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [35 S]GTPγS binding revealed a lower apparent B max value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [35 S]GTPγS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the μ-opioid receptor in brain. 相似文献
5.
Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [35 S]isethionate (10–20 pmol/g brain wet weight) and [35 S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [35 S]taurine. Taurine also produced isethionate in rat brain homogenates (about 20 nmol/h/g protein) and subcellular fractions (about 40 nmol/h/g protein in synaptosomes and about 300 nmol/h/g in mitochondria), but the reaction was not stimulated either by external electrical pulses or by the addition of various cofactors (NAD and NADP in both oxidized and reduced forms, riboflavin, glutathione. pyridoxal-5'-phosphate, ATP) to the incubation medium. [14 C]GABA was readily metabolized to [14 C]succinate both in vivo and in vitro. Isethionate formation activity was concentrated in the mitochondrial fraction, as was also GABA-T activity. Partially purified GABA-T from calf brain also slightly catalysed the formation of [35 S]isethionate (about 1.3 μmol/min/g protein) from [35 S]taurine. It appears that the slight formation of isethionate from taurine is coupled to GABA-T activity. The formation of isethionate from taurine is so small, that it apparently has no role in the control of the brain taurine pool. 相似文献
6.
Ruth A. Lanius ‡Christopher A. Shaw †Ravenska Wagey † Charles Krieger 《Journal of neurochemistry》1994,63(1):155-160
Abstract: We have characterized a high-affinity [35 S]-glutathione ([35 S]GSH) binding site in mouse and human spinal cord. [35 S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High-affinity [35 S]GSH binding was saturable, showing a B max of 72 fmol/mg of protein and a K D of 3.0 n M for mouse spinal cord and a B max of 52 fmol/mg of protein and a K D of 1.6 n M for human spinal cord. [35 S]GSH binding was displaceable by GSH, l -cysteine, and S -hexyl-GSH, but not by glutamate, glycine, or NMDA. These [35 S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [35 S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13-diacetate for 1 h before ligand binding. Phorbol ester treatment increased [35 S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a general protein kinase inhibitor. [35 S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS. 相似文献
7.
J. A. Sturman 《Journal of neurochemistry》1979,32(3):811-816
Abstract— The fate of [35 S]taurine injected intraperitoneally or intracranially has been compared in rats throughout neonatal development. The amount of [35 S]taurine present in the whole rat pup 24 h after intraperitoneal injection increases during development to a maximum 15 days after birth, and declines thereafter, whereas the amount of [35 S]taurine reaching the brain 24 h after intraperitoneal injection was greatest in the first 5 days after birth. The amount of [35 S]taurine remaining in the brain 24 h after intracranial injection does not vary throughout the period of neonatal development. These results suggest that the 'blood-brain' barrier is more accessible to taurine in the first few days after birth than later in neonatal development, and that factors other than simple exchange are involved. 相似文献
8.
Carme Solà Emili Martínez Lluïsa Camón Angel Pazos† Eduard Rodríguez-Farré 《Journal of neurochemistry》1993,60(5):1821-1834
Abstract: The effect of lindane administration on the specific binding of ligands to different sites on the GABAA receptor-ionophore complex was studied in the rat brain by receptor mapping autoradiography. [3 H]Muscimol (Mus), [3 H]flunitrazepam (Flu), and t -[35 S]butylbicyclophosphorothionate (TBPS) were used as specific ligands of GABA, benzodiazepine, and picrotoxinin binding sites, respectively. Rats received a single oral dose of 30 mg/kg lindane and they were classified into two groups according to the absence or presence of convulsions. Vehicle-treated groups acted as controls. The effect of the xenobiotic on ligand binding was measured in different brain areas and nuclei 12 min or 5 h after its administration. Lindane induced a generalized decrease in [35 S]TBPS binding, which was present shortly after dosing. In addition, [3 H]Flu binding was increased in lindane-treated animals, this modification also appearing shortly after administration but diminishing during the studied time. Finally, lindane induced a decrease in [3 H]Mus binding, which became more evident over time. These modifications were observed both in the presence and in the absence of convulsions. However, an increase in [3 H]-Mus binding was detected shortly after lindane-induced convulsions. The observed decrease in [35 S]TBPS binding is in agreement with the postulated action of lindane at the picrotoxinin binding site of the GABAA receptor chloride channel. The effects observed on the binding of [3 H]Flu and [3 H]Mus may be secondary to the action of lindane as an allosteric antagonist of the GABAA receptor. 相似文献
9.
Delphine Beaujean Ayikoe G. Mensah-Nyagan Jean-Luc Do-Rego Van Luu-The Georges Pelletier Hubert Vaudry 《Journal of neurochemistry》1999,72(2):848-857
Abstract : Biosynthesis of the neuroactive steroids pregnenolone sulfate (▵5 PS) and dehydroepiandrosterone sulfate (DHEAS) is catalyzed by the enzyme hydroxysteroid sulfotransferase (HST), which transfers the sulfonate moiety from 3'-phosphoadenosine 5' -phosphosulfate (PAPS) on thye 3-hydroxy site of steroids. Although high concentrations of ▵5 PS and DHEAS have been detected in the rat brain, the anatomical localization of HST in the CNS has never been determined. Using an antiserum against rat liver HST, we have investigated the distribution of HST-like Immunoreactivity in the CNS of the frog Rana ridibunda. Two populations of HST-immunoreactive neurons were observed in the hypothalamus, and several bundless of positive nerves fibers were visualized in the telencephalon and diencephalon. lncubation of frog brain homogenates with [35 S]PAPS and [3 H] pregnenolone yielded the formation of several 3 H, 35 S-labeled compounds, including ▵5 PS and testosterone sulfate. When [3 ] dehydroepiandrosterone and [35 S]PAPS were used as precursors, one of the 3 H, 35 S-labeled metabolities coeluted with DHEAS. Neosynthesis of [3 H]▵5 PS and [3 H]DHEAS was reduced significantly by 2,4-dichloro-6-nitrophenol, a specific inhibitor of sulfotransferases. The present study provides the first immunocytochemical mapping of HSt in the brain. Our data also demonstrate for the first time that biopsynthesis of the highly poten neuroactive steroids ▵5 PS and DHEAS occurs in the CNS of nonmammalian vertebrates. 相似文献
10.
Abstract: In membranes of rat olfactory bulb, a brain region in which muscarinic agonists increase cyclic AMP formation, the muscarinic stimulation of guanosine 5'- O -(3-[35 S]thiotriphosphate) ([35 S]GTPγS) binding was used as a tool to investigate the receptor interaction with the guanine nucleotide-binding regulatory proteins (G proteins). The stimulation of the radioligand binding by carbachol (CCh) was optimal (threefold increase) in the presence of micromolar concentrations of GDP and 100 m M NaCl. Exposure to N -ethylmaleimide and pertussis toxin markedly inhibited the CCh effect, whereas it increased the relative stimulation of [35 S]GTPγS binding elicited by pituitary adenylate cyclase-activating polypeptide (PACAP). On the other hand, membrane treatment with cholera toxin curtailed the PACAP stimulation of [35 S]GTPγS binding but did not affect the response to CCh. Like CCh, a number of cholinergic agonists stimulated [35 S]GTPγS binding in a concentration-dependent and saturable manner. The antagonist profile of the muscarinic stimulation of [35 S]GTPγS binding was highly correlated with that displayed by the muscarinic stimulation of adenylyl cyclase. These data indicate that the olfactory bulb muscarinic receptors couple to Gi /Go , but not to Gs , and support the possibility that activation of Gi /Go mediates the stimulatory effect on adenylyl cyclase activity. 相似文献
11.
The Effect of Colchicine and Cytochalasin B on the Release of Taurine from the Chick Retina 总被引:1,自引:1,他引:0
Abstract: The effect of colchicine (0.5 mM) and of cytochalasin B (10−4 M) on the release of [35 S]taurine from the isolated chick retina, upon stimulation by 68.5 mM-KCl, 10−5 M-veratridine and 10 mM-glutamate, was studied. Cytochalasin and colchicine effects on taurine release were compared with those on K+ -stimulated release of [3 H]dopamine and [3 H]GABA. Colchicine caused a marked decrease of the [35 S]taurine release evoked by the three stimulatory agents; it also decreased [3 H]dopamine release without affecting that of [3 H]GABA. Cytochalasin B significantly decreased the efflux of [35 S]taurine stimulated by glutamate or veratridine without altering that evoked by 68.5 mM-KCl. Cytochalasin practically suppressed the [3 H]dopamine-stimulated release and slightly decreased that of [3 H]GABA. This drug produced a high increase in the spontaneous release of labeled GABA and taurine. These results suggest that the release of taurine and GABA from the chick retina probably occurs through different mechanisms. It is suggested that taurine release may be related to a process involving contractile proteins. 相似文献
12.
Abstract— Brain RNP particles were characterized to determine whether they play a role in the regulation of brain protein synthesis. RNP particles were isolated from the postribosomal supernatant of cerebral hemispheres of young rabbits, employing conditions which minimize adventitious protein-RNA interactions. Brain RNP particles consist of a different set of proteins compared to proteins associated with either 40 and 60s ribosomal subunits or polysomal mRNA. Poly(A+)mRNA from brain RNP particles stimulates the incorporation of [35 S]methionine in a wheat embryo cell-free system and codes for a different set of proteins compared to poly(A+)mRNA isolated from polysomes (with some overlap; i.e. mRNA coding for brain-specific S100 protein is present in both RNP particles and polysomes).
Addition of total brain RNP particles to a cell-free wheat embryo system inhibits the endogenous incorporation of [35 S]methionine. Total RNP particles were fractionated by sucrose density gradient centrifugation into a'light'and a'heavy'fraction. The light RNP fraction inhibited while the heavy RNP fraction stimulated protein synthesis in the wheat embryo cell-free system. Analysis of the protein composition of fractionated RNP particles revealed that the light and heavy RNP particles contained different sets of proteins. Together these results suggested that one class of brain RNP particles may contain a translational inhibitor and may be involved in the regulation of protein synthesis in the brain. 相似文献
Addition of total brain RNP particles to a cell-free wheat embryo system inhibits the endogenous incorporation of [
13.
UPTAKE AND RELEASE OF TAURINE FROM RAT BRAIN SLICES 总被引:13,自引:8,他引:5
Abstract— Rapid efflux of [35 S]taurine from rat brain slices was observed on electrical stimulation. Slower release resulted when the Ca2+ content of the perfusion medium was replaced with Mg2+ . Uptake of [35 S]taurine into rat cortical slices was unaffected by GABA, glutamic acid, glycine and leucine but was inhibited by alanine, ouabain, KCN and 2,4-dinitrophenol. Of a number of analogues of taurine, 2-aminoethylsulphinic acid was the most potent in inhibiting the uptake of [35 S]taurine. The rate of uptake was found to be decreased by lowering the incubation temperature. The possibility that taurine may be a neurotransmitter is discussed. 相似文献
14.
15.
Biosynthesis of Polyamines in Mouse Brain: Effects of Methionine Sulfoximine and Adenosylhomocysteine 总被引:1,自引:1,他引:0
Raffaele Porta Robert A. Schatz Stephen B. Tatter Otto Z. Sellinger 《Journal of neurochemistry》1983,40(3):836-841
Abstract: This study examines the consequences on cerebral polyamine biosynthesis of increases and decreases in cerebral methylation. Increases were elicited by administering the convulsant agent methionine sulfoximine (MSO) and decreases by elevating in vivo the cerebral levels of the methylation inhibitor S -adenosyl-homocysteine. Following the intraventricular (i.vt.) administration of one of the two possible polyamine precursors, [1,4-14 C]putrescine, the specific radioactivity (sra) of the newly formed [14 C]spermidine remained unchanged. Conversely, after i.vt. l -[3,4-14 C]methionine, the other polyamine precursor, significantly higher sra values for [14 C]spermidine and [14 C]spermine were recorded in the brains of the MSO-treated animals. [14 C] S - adenosylmethionine in the brain of the MSO-treated animals was also more highly labeled following [1-14 C]-methionine, indicating its accelerated formation relative to controls. We also investigated the effect of the administration of adenosine + homocysteine, a treatment that results in elevated brain adenosylhomocysteine levels, on polyamine biosynthesis from [3,4-14 C]-methionine. The results of these experiments show both significantly lower sra values for [14 C]spermidine and [14 C]spermine and significantly higher than control endogenous methionine levels, a clear sign of the existence of a retardation in the conversion of methionine to polyamines under these conditions. In conclusion, the present study demonstrates that while interference with cerebral methylation results in significant alterations of the rate of formation of the methionine moiety of spermidine and spermine, it has no effect on the entry of the putrescine moiety into the two polyamine molecules. 相似文献
16.
n-3 Fatty Acid Deficiency Increases Brain Protein Synthesis in the Free-Moving Adult Rat 总被引:1,自引:0,他引:1
Murielle Giaume Nadine Gay Valérie Baubet †Abdallah Gharib ‡Georges Durand Pierre Bobillier † Nicole Sarda 《Journal of neurochemistry》1994,63(5):1995-1998
Abstract: The autoradiographic method with l -[35 S]methionine was used to determine the effects of an n-3 fatty acid deficiency on brain protein synthesis. Brain protein synthesis was significantly increased (from 50 to 150%) in 45 of the 52 brain structures studied in n-3 fatty acid-deficient rats as compared with control animals. Biochemical analysis confirmed the increase in overall rate of protein synthesis in brain as a whole. 相似文献
17.
MARIA E. HOFFMANN JUNHYUK JANG SILVIA N. J. MORENO ROBERTO DOCAMPO 《The Journal of eukaryotic microbiology》1995,42(3):293-297
ABSTRACT. [35 S]methionine incorporation into proteins of either T. cruzi epimastigotes or trypomastigotes was drastically inhibited by low concentrations of crystal violet in a dose-dependent manner. This inhibition was not due to ATP depletion since cellular ATP levels did not change significantly after incubation of epimastigotes with 50 μM crystal violet for similar periods of time, and was unaffected by changes in the extracellular free calcium concentration. Although crystal violet was able to inhibit protein synthesis in a cell-free system from T. cruzi epimastigotes, half maximal inhibition was at 1 mM, a concentration three orders of magnitude higher than those that inhibited protein synthesis in intact cells. On the other hand, crystal violet was able to inhibit total [35 S]methionine uptake at similar concentrations to those that inhibited protein synthesis while addition of increasing concentrations of cold methionine to the incubation medium protected the cells against crystal violet inhibition. Crystal violet also inhibited total [3 H]proline uptake thus indicating that it has a general inhibitory effect upon the transport of amino acids, and not specifically upon methionine. These results indicate that inhibition of protein synthesis by crystal violet is probably due to inhibition of amino acid uptake. 相似文献
18.
Christian Chabert Catherine Cavegn Alain Bernard Ann Mills 《Journal of neurochemistry》1994,63(1):62-65
Abstract: The human D4 dopamine receptor has been expressed in Sf9 insect cells where it appears to couple to endogenous G proteins. Increased guanine nucleotide exchange to G proteins is a reflection of receptor activation and can be followed using a [35 S]GTPγS binding assay. By measuring D4 receptor stimulation of [35 S]-GTPγS binding we have been able to characterize several dopaminergic compounds for their functional activity at this receptor. In Sf9 cells expressing the D4 receptor, dopamine, quinpirole, and dp -2-aminodihydroxy-1,2,3,4-tetrahydronaphthalene were all full agonists, whereas (−)-apomorphine appeared to be a partial agonist. No increase in [35 S]GTPγS binding was observed for noninfected cells or cells infected with an unrelated sequence. The quinpirole-stimulated [35 S]GTPγS binding could be inhibited by the antagonists clozapine, eticlopride, and haloperidol, and a Schild analysis of these data showed that all three compounds were acting as competitive antagonists of D4 receptors. The rank order of affinities derived from the Schild analysis correlated with that obtained from [3 H]spiperone competition binding assays. In conclusion, we have shown that, using this assay system, it is possible to investigate functionally the pharmacology of a recombinant G protein-coupled receptor in the absence of any information regarding the eventual second messenger pathways involved. 相似文献
19.
Reynold Spector 《Journal of neurochemistry》1981,36(6):2019-2024
Abstract: The development of the thymidine phosphorylating systems was studied in various regions of brain. Brain slices from cerebellum, brain stem, and forebrain of rabbits 2, 7, 14, 30, 90, 500, and 2500 days of age were incubated for various times in artificial CSF containing 3 nM-[3 H]thymidine at 37°C under 95% O2 -5% CO2 . When slices from all brain regions of 2-day-old rabbits were incubated in [3 H]thymidine for 30 min, tissue-to-medium ratios of 3 H were between 2 and 4 and declined with age, and the percentages of the total 3 H in perchloric acid homogenates of brain slices as [3 H]DNA were 26–29%, declining to low levels with age. However, at all ages and in all regions studied, 41 -88% of the 3 H within the slices was phosphorylated. After homogenization and subcellular fractionation of the brain slices incubated in [3 H]thymidine for 30 min, the highest percentage of [3 H]thymidine phosphates plus [3 H]DNA was present in the nuclear (crude and purified) and mitochondrial fractions of all brain regions. The [3 H]DNA content in the nuclear and mitochondrial fractions declined with age, but the percentage of [3 H]thymidine phosphates did not. Thymidine phosphates were synthesized from thymidine in all brain regions tested throughout the entire life span. 相似文献
20.
Abstract— [35 S]cysteine, [3 H]methionine, or [3 H]fucose were injected into the supraoptic nuclei (SON) of rats, and the labelled proteins that were transported to and accumulated in the posterior pituitary 24h post-injection were analyzed electrophoretically. The transported, labelled proteins which were soluble in 0.1 m -HCl were primarily of low molecular weight (about 12,000 on SDS gels). However, the selectivity of labelling of these proteins by the three different labelled precursors could be revealed by isoelectric focusing. The 0.1 m -HCl insoluble labelled proteins, presumably reflecting membrane proteins transported from the SON to the pituitary, were more diverse and generally of higher molecular weight (> 43,000 on SDS gels). 相似文献