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  • 1
    Electronic Resource
    Electronic Resource
    Developmental Changes in the Differential Expression of Two Serotonin 5-HT3 Receptor Splice Variants in the Rat (1995)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 65 (1995), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: PCR was used to isolate identical partial cDNA clones encoding a serotonin 5-HT3 receptor subunit from rat nodose and superior cervical ganglia. The amino acid sequence predicted from these clones, extending from the putative transmembrane domain I to the stop codon, demonstrated a 93% homology with the 5-HT3 receptor A (R-A) subunit cloned from NCB 20 hybridoma mouse neuroblastoma/Chinese hamster embryonic brain cells. Comparison of the sequences of the rat gene and cDNA encoding this subunit revealed a five amino acid deletion, GSLLP, located within the putative second intracellular loop of the receptor subunit. This deletion was shown to occur at an intron/exon junction. Therefore, alternative splicing was probably responsible for the presence of short (5-HT3 R-AS) and long (5-HT3 R-AL) forms of 5-HT3 R-A mRNA in these ganglia. PCR experiments, with specific primers located upstream and downstream of the GSLLP deletion, were used to detect reverse transcribed 5-HT3 R-A mRNAs. A short fragment (92 bp), corresponding to the deleted form, and a long fragment (107 bp), corresponding to the nondeleted form, were amplified from various regions of the CNS and peripheral ganglia of the rat, as well as from NG108-15 hybridoma cells. In the adult rat, the ratio of the two forms varied very little from one tissue to another, the long form corresponding to only ∼10% of the total 5-HT3 R-A mRNA. Study of their respective distributions during ontogeny demonstrated a differential expression of the short and long forms in some tissues during late embryonic development, at embryonic day 17 (E17) or E20. In particular, the long form amounted to about one-third of the total 5-HT3 R-A mRNA in the cerebral cortex and hippocampus at E17, and this proportion reached 50 and 75% in the superior cervical ganglion and nodose ganglion, respectively, at E20. These data indicate that alternative splicing of the 5-HT3 R-A mRNA is regulated in the CNS and PNS during development in the rat.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1995.65020475.x
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  • 2
    Electronic Resource
    Electronic Resource
    Identification of the 5-HT1A Receptor Binding Subunit in Rat Brain Membranes Using the Photoaffinity Probe [3H]8-Methoxy-2-[N-n-Propyl, N-3-(2-Nitro-4-Azidophenyl)Aminopropyl]Aminotetralin (1987)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 49 (1987), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: The synthesis of a tritiated derivative of the 5-HT1A photoaffinity probe 8-methoxy-2-[N-n-propyl, N-3-(2-nitro-4-azidophenyl)aminopropyl]aminotetralin ([3H]8-methoxy-3′-NAP-amino-PAT) allowed the use of this probe for attempting the irreversible labeling of specific binding sites in rat brain membranes. Sodium dodecyl-sulfate-polyacrylamide gel electrophoresis of proteins solubilized from hippocampal microsomal membranes that had been incubated with 20 nM [3H]8-methoxy-3′-NAP-amino-PAT under UV light revealed a marked incorporation of 3H label into a 63-kilodalton protein termed PI . As expected of a possible correspondence between P1 and 5-HT1A receptor binding sites, 3H labeling by the photoaffinity probe could be prevented by selective 5-HT1A ligands such as 8-hydroxy-2-(di-n-propylamino)tetralin, ipsapirone, buspirone, and gepirone and by N-ethylmaleimide, but not by the 5-HT2 antagonist ketanserin, noradrenaline-and dopamine-related drugs, monoamine oxidase inhibitors, and chlorimipramine. Furthermore, the regional and subcellular distributions of P1 were identical to those of specific 5-HT1A binding sites. These results indicated that the binding subunit of the 5-HT1A receptor is a 63-kilodalton protein with a functionally important sulfhydryl group(s).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1987.tb02875.x
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  • 3
    Electronic Resource
    Electronic Resource
    Presynaptic Dopamine Autoreceptors Control Tyrosine Hydroxylase Activation in Depolarized Striatal Dopaminergic Terminals (1986)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 46 (1986), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The possible control of tyrosine hydroxylase (TH) activity by dopaminergic receptor-dependent mechanisms was investigated using rat striatal slices or synaptosomes incubated in the presence of various 3,4-dihydroxyphenylethylamine (dopamine or DA) agonists and antagonists. Under “normal” conditions (4.8 mM K+ in the incubating medium), the DA agonists apomorphine, 6,7-dihydroxy-N, N-dimethyl-2-aminotetralin (TL-99), 7-hydroxy-N, N-dipropyl-2-aminotetralin (7-OH-DPAT), trans- (–)-4,4a,5,6,7,8,8a,9-octahydro- 5 -propyl-2H-pyrazolo-3,4-quinoline, and 3-(3-hydroxyphenyl)-N-n-propylpiperidine decreased TH activity in soluble extracts of incubated tissues. In the case of the catechol-containing drugs apomorphine and TL-99, this effect was partly due to a direct inhibition of the enzyme, but in all other cases it appeared to depend on the stimulation of presynaptic DA autoreceptors. No effect of DA antagonists was detected on TH activity under “normal” conditions. In contrast, when tissues were incubated in a K+-enriched (60 mM) medium, (–)-sulpiride and other DA antagonists enhanced TH activation due to depolarization whereas DA agonists were ineffective. Because (–)-sulpiride also increased the enzyme activity in striatal slices exposed to drugs inducing release of DA, such as veratridine and d-amphetamine, it is concluded that the stimulating effect of the DA antagonist resulted in fact from the blockade of the negative control of TH normally triggered by endogenous DA acting on presynaptic autoreceptors. In contrast to TH activation due to K+-induced depolarization, the activation evoked by tissue incubation with dibutyryl cyclic AMP was unaffected by the typical agonist 7-OH-DPAT or the antagonist (–)-sulpiride. This would suggest that TH control via presynaptic DA autoreceptors normally concerns possible modulations of the cyclic AMP-dependent phosphorylation of the enzyme.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1986.tb12919.x
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  • 4
    Electronic Resource
    Electronic Resource
    Differentiation Alters the Expression of the Two Splice Variants of the Serotonin 5-HT3 Receptor-A mRNA in NG108-15 Cells (1995)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 65 (1995), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The serotonin 5-HT3-A receptor (5-HT3R-A) mRNA has been shown recently to be expressed as two forms (5-HT3R-AL and 5-HT3R-AS) varying by the presence or the absence of a sequence of 18 bases in the region corresponding to the second cytoplasmic domain of the receptor, and generated by alternative splicing at the level of the 3′ acceptor site of exon 9. As the long form of the receptor exhibits a potential phosphorylation site that is disrupted by the alternative splicing, the hypothesis of functional identity and stochastic expression of these two variants was questioned. In the present study, we used quantitative reverse transcriptase-polymerase chain reaction to examine the possible influence of culture conditions on the expression and the alternative splicing of 5-HT3R-A mRNA in NG108-15 clonal cells. Cell differentiation induced by dibutyryl cyclic AMP or theophyllin plus prostaglandin E1 in the presence of 10% serum reduced by threefold the expression of total 5-HT3R-A mRNA, and favored the short form of the message as the ratio S/L (5-HT3R-AS mRNA/5-HT3R-AL mRNA) shifted from 2.23 to 7.33 after 9 days of treatment. Culture with 0.3% serum (instead of 10%) lowered by 10-fold the level of expression of total 5-HT3R-A mRNA, but only slightly reduced the S/L ratio. However, this ratio fell to 0.06 in the presence of 0.3% serum plus 10 ng/ml basic fibroblast growth factor. These results demonstrate that external factors can influence the differential expression of the two variants of the 5-HT3R-A in NG108-15 cells. Appropriate culture conditions for the almost exclusive expression of 5-HT3R-AS mRNA or 5-HT3R-AL mRNA in NG108-15 cells should allow the identification of possible differences in the respective functional properties of each of these two forms of the native 5-HT3 receptor.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1995.65051917.x
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  • 5
    Electronic Resource
    Electronic Resource
    Chromatographic Analyses of the Serotonin 5-HT1A Receptor Solubilized from the Rat Hippocampus (1989)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 53 (1989), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Serotonin 5-HT1A receptors in rat hippocampal membranes were solubilized by 10 mM 3-[3-(cholamidopro-pyl)dimethylammonio]-l-propane sulfonate (CHAPS) and chromatographed on various gels in an attempt to design a relevant protocol for their (partial) purification. In particular, an affinity gel made of the 8-hydroxy-2-(di-n-propylami-no)tetralin (8-OH-DPAT) derivative 8-methoxy-2-[(N-propyl, N-butylamino)amino]tetralin (8-MeO-N-PBAT) coupled to Affigel 202 was specially developed for this purpose. First, studies of the effects of various compounds (detergents, lipids, reducing agents, sugars, etc.) on the specific binding of [3H]8-OH-DPAT and on the rate of heat-induced inactivation of solubilized 5-HT1A sites led to a buffer composed of 50 mM Tris-HCl, 50 μM dithiothreitol, 1 mM CHAPS, 10% glycerol, 0.1 mA/ MnCl2, and 50 μg/ml of cholesleryl hemisuccinate, pH 7.4, ensuring a high degree of stability of solubilized 5-HT1A sites, compatible with chromatographic analyses for 2–4 days at 4°C. Adsorption and subsequent elution of [3H]8-OH-DPAT specific binding sites were found with several chromatographic gels, including wheat germ agglutinin-agarose, phenyl-Sepharose, hydroxylapatite-Ultrogel, diethyl-aminoethyl (DEAE)-Sepharose, and DEAE-Sephacel. Similarly, 8-MeO-N-PBAT-Affigel 202 allowed the adsorption and subsequent elution (by 1 mM 5-HT) of active 5-HT1Abinding sites solubilized from rat hippocampal membranes. The two-step chromatography using 8-MeO-N-PBAT-Affigel 202 followed by wheat germ agglutinin-agarose gave a fraction enriched (by at least 400-fold) in 5-HT1A sites. Sodium do-decyl sulfate-polyacrylamide gel electrophoresis of this partially purified fraction revealed a major protein band with Mr close to 60,000.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1989.tb08552.x
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  • 6
    Electronic Resource
    Electronic Resource
    Pharmacological and Biochemical Characterization of Rat Hippocampal 5-Hydroxytryptamine1A Receptors Solubilized by 3–[3–(Cholamidopropyl)dimethylammonio]-1-Propane Sulfonate (CHAPS) (1988)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 51 (1988), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Rat hippocampal 5-hydroxytryptamine1A (5-HT1A) binding sites were solubilized with a yield of 34% using 3–[3–(cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS, 10 mM) as detergent. Kinetic analyses of [3H]8-hydroxy-2–(di-n-propylamino)tetralin ([3H]8-OH-DPAT) binding indicated that the 5-HT1A sites exhibit the same properties in the soluble form as in the membrane-bound form. Furthermore, a positive correlation (r= 0.988) was found between the respective pIC50 values of a series of agonists and antagonists to inhibit [3H]8-OH-DPAT binding to either soluble or membrane-bound 5-HT1A sites. Gel filtration through Sephacryl S-400 as well as chromatography on wheat germ agglutinin (WGA)-agarose did not affect the modulation by guanine nucleotides (5′-guanylylimidodi-phosphate) of [3H]8-OH-DPAT binding which suggests that the 5-HT1A binding subunit is a glycoprotein tightly attached to a G protein even in its soluble form. The [3H]8-OH-DPAT binding material eluted from Sephacryl S-400 had an apparent molecular mass of 155 kilodaltons, as expected from a heterodimer with one binding subunit (∼60 kilodaltons) and one G protein (∼80 kilodaltons). Marked enrichment in 5-HT1A binding sites relative to other soluble proteins was found in the peak fractions eluted from Sephacryl S-400 (by sixfold) and WGA-agarose (by 26-fold) columns, suggesting that these chromatographic steps might be of interest for the purification of central 5–HT1A receptors.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1988.tb03064.x
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  • 7
    Electronic Resource
    Electronic Resource
    Immunolabeling of Central Serotonin 5-HT1Dβ Receptors in the Rat, Mouse, and Guinea Pig with a Specific Anti-Peptide Antiserum (1995)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 65 (1995), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5-HT1B/1Dβ receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti-peptide antibodies were detected by enzyme-linked immunosorbent assay and characterized by immunoprecipitation of the 5-HT1B/1Dβ receptor in CHAPS-solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin-O-carboxymethylglycyl[125I]iodotyrosinamide ([125I]GTI; a selective 5-HT1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5-HT1B receptors. The remaining 40% of [125I]GTI binding sites were shown to be 5-HT1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5-HT1B/1Dβ, but not the 5-HT1D/1Dα, receptor in transiently transfected LLC-PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5-HT1Dβ receptor-like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [125I]GTI in the brain of these species further confirmed that the anti-peptide antibodies selectively recognized only the 5-HT1Dβ component of [125I]GTI specific receptor binding sites.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1995.65062671.x
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  • 8
    Electronic Resource
    Electronic Resource
    Is Dopamine-Induced Inhibition of Adenylate Cyclase Involved in the Autoreceptor-Mediated Negative Control of Tyrosine Hydroxylase in Striatal Dopaminergic Terminals? (1986)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 47 (1986), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract The mechanism of the negative control of tyrosine hydroxylase (TH) activity induced by the stimulation of presynaptic 3,4-dihydroxyphenylethylamine (dopamine, DA) autoreceptors was investigated using rat striatal slices and synaptosomes incubated under control ([K+] = 4.8 mM) or depolarizing ([K+] = 60 mM) conditions. The stimulation of DA autoreceptors by 7-hydroxy-2-(di-n-propylamino) tetralin (1 μM 7-OH-DPAT) produced a significant decrease in TH activity extracted from striatal slices maintained under control conditions. This effect was associated with the complete conversion of TH into an enzyme form with a low affinity for its pterin cofactor (Km∼0.80 mM). Furthermore, compared to TH extracted from control tissues, that from 7-OH-DPAT-exposed striatal slices was more sensitive to the stimulatdry effects of exogenous heparin and cyclic AMP-dependent phosphorylation. Such changes were opposite to those induced by incubating striatal slices with the adenylate cyclase activator forskolin. Indeed, forskolin treatment completely converted TH into an enzyme form with a high affinity for its pterin cofactor (Km∼0.16 mM). Such conversion was associated with a shift in the optimal pH for TH activity from 5.8 (control) to 7.2 (forskolin). Under depolarizing conditions, the blockade by (—)-sulpiride of the stimulation of DA autoreceptors by endogenous DA was associated with a marked activation of TH. Modifications of enzymatic characteristics triggered by (—)-sulpiride were then similar to those induced by forskolin treatment. These data suggest that presynaptic DA autoreceptors modulate the activity of TH by controlling the degree of cyclic AMP-dependent phosphorylation of the enzyme. The blockade by Pertussis toxin of the 7-OH-DPAT-induced inhibition of TH activity is coherent with a possible negative coupling of presynaptic DA autoreceptors (closely related to the D2 type) with adenylate cyclase. Such negative coupling would account for the reduction of TH activity when presynaptic DA autoreceptors are stimulated.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1986.tb00775.x
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  • 9
    Electronic Resource
    Electronic Resource
    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 (1982)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 38 (1982), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: 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 (KD= 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 nucleotide2- 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1982.tb10867.x
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  • 10
    Electronic Resource
    Electronic Resource
    The “Orphan” Na+/Cl--Dependent Transporter, Rxt1, Is Primarily Localized Within Nerve Endings of Cortical Origin in the Rat Striatum (1999)
    Oxford UK : Blackwell Science Ltd.
    Journal of neurochemistry 73 (1999), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract : Previous studies have shown that the striatum expresses very low levels of Na+/Cl--dependent “orphan” transporter Rxt1 transcripts but contains high levels of protein. This study investigated the origin of Rxt1 expression in rat striatum. Striatal Rxt1 contents assessed by immunocytochemistry or western blotting were found to be significantly reduced after corticostriatal denervation but not after striatal or thalamic lesion with kainic acid or selective 6-hydroxydopamine-induced nigrostriatal deafferentation. Corticostriatal neurons retrogradely labeled by intrastriatal fluorogold injections were shown to express Rxt1 mRNA. Combination of anterograde biotin-dextran amine labeling of the corticostriatal pathway with Rxt1 immunogold detection at the ultrastructural level demonstrated the presence of Rxt1 in about one-third of the corticostriatal synaptic terminals and in numerous unidentified synaptic terminals. All the Rxt1-positive terminals formed asymmetrical contacts on spines. These data provide evidence that striatal Rxt1 immunoreactivity is mainly of extrinsic origin and more specifically associated with the corticostriatal pathway. Rxt1 appears as a selective presynaptic marker of synapses formed by presumably excitatory amino acid afferents, but it segregates a subclass of these synapses, thereby revealing a functional heterogeneity among excitatory amino acid systems.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1999.0730623.x
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