RESUMEN
Serotonin (5-hydroxytryptamine; 5-HT) signaling through the 5-HT(2C) receptor (5-HT(2C)R) is essential in normal physiology, whereas aberrant 5-HT(2C)R function is thought to contribute to the pathogenesis of multiple neural disorders. The 5-HT(2C)R interacts with specific protein partners, but the impact of such interactions on 5-HT(2C)R function is poorly understood. Here, we report convergent cellular and behavioral data that the interaction between the 5-HT(2C)R and protein phosphatase and tensin homolog (PTEN) serves as a regulatory mechanism to control 5-HT(2C)R-mediated biology but not that of the closely homologous 5-HT(2A)R. A peptide derived from the third intracellular loop of the human 5-HT(2C)R [3L4F (third loop, fourth fragment)] disrupted the association, allosterically augmented 5-HT(2C)R-mediated signaling in live cells, and acted as a positive allosteric modulator in rats in vivo. We identified the critical residues within an 8 aa fragment of the 3L4F peptide that maintained efficacy (within the picomolar range) in live cells similar to that of the 3L4F peptide. Last, molecular modeling identified key structural features and potential interaction sites of the active 3L4F peptides against PTEN. These compelling data demonstrate the specificity and importance of this protein assembly in cellular events and behaviors mediated by 5-HT(2C)R signaling and provide a chemical guidepost to the future development of drug-like peptide or small-molecule inhibitors as neuroprobes to study 5-HT(2C)R allostery and therapeutics for 5-HT(2C)R-mediated disorders.
Asunto(s)
Modelos Moleculares , Fosfohidrolasa PTEN/química , Fosfohidrolasa PTEN/metabolismo , Receptor de Serotonina 5-HT2C/química , Receptor de Serotonina 5-HT2C/metabolismo , Transducción de Señal/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Immunoblotting , Inmunoprecipitación , Masculino , Datos de Secuencia Molecular , Actividad Motora/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Ratas , Ratas Sprague-Dawley , Serotonina/metabolismo , TransfecciónRESUMEN
BACKGROUND: The serotonin (5-HT) 2A and 2C receptors (5-HT2AR and 5-HT2CR) are involved in a wide range of physiological and behavioral processes in the mammalian central and peripheral nervous systems. These receptors share a high degree of homology, have overlapping pharmacological profiles, and utilize many of the same and richly diverse second messenger signaling systems. We have developed quantitative assays for cells stably expressing these two receptors involving minimal cell sample manipulations that dramatically improve parallel assessments of two signaling responses: intracellular calcium (Cai++) changes and activation (phosphorylation) of downstream kinases. Such profiles are needed to begin to understand the simultaneous contributions from the multiplicity of signaling cascades likely to be initiated by serotonergic ligands. RESULTS: We optimized the Cai++ assay for stable cell lines expressing either 5-HT2AR or 5-HT2CR (including dye use and measurement parameters; cell density and serum requirements). We adapted a quantitative 96-well plate immunoassay for pERK in the same cell lines. Similar cell density optima and time courses were observed for 5-HT2AR- and 5-HT2CR-expressing cells in generating both types of signaling. Both cell lines also require serum-free preincubation for maximal agonist responses in the pERK assay. However, 5-HT2AR-expressing cells showed significant release of Cai++ in response to 5-HT stimulation even when preincubated in serum-replete medium, while the response was completely eliminated by serum in 5-HT2CR-expressing cells. Response to another serotonergic ligand (DOI) was eliminated by serum-replete preincubation in both cells lines. CONCLUSIONS: These data expand our knowledge of differences in ligand-stimulated signaling cascades between 5-HT2AR and 5-HT2CR. Our parallel assays can be applied to other cell and receptor systems for monitoring and dissecting concurrent signaling responses.
Asunto(s)
Calcio/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Receptor de Serotonina 5-HT2A/metabolismo , Receptor de Serotonina 5-HT2C/metabolismo , Agonistas de Receptores de Serotonina/farmacología , Animales , Células CHO , Señalización del Calcio/fisiología , Cricetinae , Medios de Cultivo , Humanos , Ligandos , Fosforilación/efectos de los fármacos , Serotonina/farmacologíaRESUMEN
The action of serotonin (5-HT) at the 5-HT(2C) receptor (5-HT(2C)R) in cerebral cortex is emerging as a candidate modulator of neural processes that mediate core phenotypic facets of several psychiatric and neurological disorders. However, our understanding of the neurobiology of the cortical 5-HT(2C)R protein complex is currently limited. The goal of the present study was to explore the subcellular localization of the 5-HT(2C)R in synaptosomes and the post-synaptic density, an electron-dense thickening specialized for post-synaptic signaling and neuronal plasticity. Utilizing multiples tissues (brain, peripheral tissues), protein fractions (synaptosomal, post-synaptic density), and controls (peptide neutralization, 5-HT(2C)R stably-expressing cells), we established the selectivity of two commercially available 5-HT(2C)R antibodies and employed the antibodies in western blot and immunoprecipitation studies of prefrontal cortex (PFC) and motor cortex, two regions implicated in cognitive, emotional and motor dysfunction. For the first time, we demonstrated the expression of the 5-HT(2C)R in post-synaptic density-enriched fractions from both PFC and motor cortex. Co-immunoprecipitation studies revealed the presence of post-synaptic density-95 within the 5-HT(2C)R protein complex expressed in PFC and motor cortex. Taken together, these data support the hypothesis that the 5-HT(2C)R is localized within the post-synaptic thickening of synapses and is therefore positioned to directly modulate synaptic plasticity in cortical neurons.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Corteza Motora/citología , Neuronas/ultraestructura , Receptor de Serotonina 5-HT2C/metabolismo , Sinaptosomas/metabolismo , Animales , Técnicas de Cultivo de Célula , Cricetinae , Cricetulus , Regulación de la Expresión Génica/genética , Inmunoprecipitación/métodos , Masculino , Modelos Moleculares , Peso Molecular , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2C/química , Receptor de Serotonina 5-HT2C/genética , Transfección/métodosRESUMEN
This study investigated estradiol (E(2)) modulation of the antidepressant effects of a selective serotonin (5-HT) reuptake inhibitor (SSRI; sertraline) and a tricyclic antidepressant (imipramine) as measured by the forced swim test (FST) followed by assessment of gene and protein expression for the 5-HT transporter (SERT) and multiple 5-HT receptors. Female Sprague-Dawley rats were ovariectomized (OVX) and two-thirds of the rats received E(2) implants (OVE). 4 weeks later, implants were withdrawn in half of the OVE rats (OVW) to capture a time point when E(2) levels were rapidly declining. Rats in each hormone group were treated with vehicle, sertraline (10 mg/kg) or imipramine (10 mg/kg), 24, 5 and 1h before the FST. Immediately after the FST, midbrain, hippocampus and prefrontal cortex tissue was removed and frozen for analysis of gene expression via quantitative real-time PCR (midbrain tissue) and protein expression via Western blot (prefrontal cortex and hippocampal tissue). In the FST, sertraline decreased immobility and increased swimming in OVE rats, as well as increased swimming in OVW rats. In contrast, no sertraline effect was observed in OVX rats. Rats treated with imipramine showed increased climbing but no changes in immobility or swimming. No changes in protein expression were detected in any treatment group. However, in vehicle-treated rats, E(2) increased midbrain SERT mRNA expression, with no effect on midbrain mRNA for the 5-HT receptors. In sertraline-treated rats, E(2) decreased 5-HT(2A) receptor mRNA, and E(2)-withdrawal increased 5-HT(1A), 5-HT(2A) and 5-HT(2C) receptor mRNA. In imipramine-treated rats, E(2) (and E(2)-withdrawal) did not affect mRNA expression for any of the target genes. Thus, E(2) synergized behaviorally and neurochemically with an SSRI but not a tricyclic antidepressant.
Asunto(s)
Depresión/tratamiento farmacológico , Estradiol/administración & dosificación , Sertralina/administración & dosificación , Animales , Antidepresivos/administración & dosificación , Antidepresivos/farmacología , Conducta Animal/efectos de los fármacos , Depresión/patología , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Estradiol/sangre , Estradiol/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Ovariectomía , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Sertralina/farmacología , Vagina/efectos de los fármacos , Vagina/metabolismo , Vagina/patologíaRESUMEN
Gamma-aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA) provide innervation to cortical and subcortical regions of the brain. To solidify the importance of these VTA GABA neurons in behavioral function, we employed the neurotoxin dermorphin-saporin (DS) to selectively lesion VTA GABA neurons prior to assessing spontaneous motor activity. Rats were bilaterally microinfused with DS (1.0 or 2.0 pmol/200 nl/side) or blank-saporin control (BS, 200 nl/side) into the VTA. Seven days later, DS-treated rats exhibited significantly elevated motility in comparison with BS-treated rats; this elevated motility normalized by Day 14 following pretreatment with 1.0 pmol of DS but was sustained on Day 14 after pretreatment with 2.0 pmol of DS. A selective loss of VTA GABA neurons on Day 14 was demonstrated through reduced expression of mRNA for glutamic acid decarboxylase-67 and micro-opioid receptor, but not tyrosine hydroxylase (a dopamine neuron marker), in the VTA. Thus, a dose- and time-related selective loss of VTA GABA neurons was accomplished using this novel neurotoxin. This loss of GABA VTA neurons was associated with hypermotility, further supporting their important regulatory role in the generation of behavior.
Asunto(s)
Actividad Motora/fisiología , Neuronas/fisiología , Área Tegmental Ventral/citología , Área Tegmental Ventral/fisiología , Ácido gamma-Aminobutírico/metabolismo , Análisis de Varianza , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Glutamato Descarboxilasa/genética , Glutamato Descarboxilasa/metabolismo , Masculino , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neurotoxinas/efectos adversos , Péptidos Opioides/efectos adversos , Ratas , Ratas Sprague-Dawley , Proteínas Inactivadoras de Ribosomas Tipo 1/efectos adversos , Saporinas , Factores de Tiempo , Tirosina 3-Monooxigenasa/metabolismo , Área Tegmental Ventral/efectos de los fármacosRESUMEN
Surface enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS) is an analytical technology for proteomic analysis that combines chromatography and mass spectrometry. At present, this technology is most commonly being exploited for the simultaneous measurement of numerous proteins in serum, but has also been utilized in organ tissue, although rarely in the brain. We applied SELDI-TOF MS technology to study protein expression in the brain of rats withdrawn from repeated cocaine exposure. Our goals were to optimize sample preparation and ProteinChip Array protocols for brain tissue, to verify the reproducibility of SELDI-TOF mass spectra and to determine whether SELDI-TOF MS detects differentially expressed proteins in cocaine- versus saline-treated rats. Consequently, we have developed an optimal protocol and generated a reproducible spectral pattern with six dominant peaks in all test samples. We have detected two smaller peaks (m/z: 5179, 5030) that were significantly increased (p < 0.05) in cocaine-treated rats compared to saline-treated rats. In summary, the application of SELDI-TOF MS to the study of protein expression in a rat model of cocaine withdrawal is feasible and has the potential to generate new hypotheses.
Asunto(s)
Cocaína/efectos adversos , Proteínas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Síndrome de Abstinencia a Sustancias/etiología , Síndrome de Abstinencia a Sustancias/metabolismo , Animales , Conducta Animal , Modelos Animales de Enfermedad , Masculino , Análisis por Matrices de Proteínas/métodos , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/metabolismoRESUMEN
Although radioligand binding studies have shown that the serotonin 5-HT(2C) receptor (5-HT(2C)R) is widely expressed throughout the brain, more detailed knowledge of 5-HT(2C)R distribution within different neuronal populations will aid in understanding the mechanisms through which this receptor acts. Double-label immunohistochemical procedures can be utilized to examine the localization of receptors within specific neuronal populations. In order to conduct such studies, however, it was first necessary to examine the utility and specificity of two commercially available anti-5-HT(2C)R antibodies [from Santa Cruz (SC) and BD PharMingen (PH)]. In male Sprague-Dawley rats, both antibodies produced widespread immunoreactivity (IR) throughout the brain area chosen for study, the ventral tegmental area, which is the origin of the dopamine mesocorticoaccumbens "reward" pathway. Co-labeling with the SC and PH 5-HT(2C)R antibodies demonstrated that IR for the two antibodies largely overlapped. However, SC 5-HT(2C)R IR was more concentrated within IR cell bodies and was more consistent among assays than the PH 5-HT(2C)R IR. Thus, the SC 5-HT(2C)R antibody was chosen for subsequent studies. When examined in 5-HT(2C)R knockout vs. wild-type mice, the SC 5-HT(2C)R antibody produced widespread IR in wild-type, but not 5-HT(2C)R knockout, mice. In addition, 5-HT(2C)R-IR was not present in either native CHO cells, known to be devoid of 5-HT(2A)R or 5-HT(2C)R, or in CHO cells transfected with the 5-HT(2A)R. Thus, these studies suggest that the SC 5-HT(2C)R antibody produces reliable staining selective for 5-HT(2C)R vs. 5-HT(2A)R in rodent brains and is therefore suitable for use in future immunofluorescence 5-HT(2C)R localization studies.
Asunto(s)
Especificidad de Anticuerpos , Técnica del Anticuerpo Fluorescente/métodos , Técnica del Anticuerpo Fluorescente/normas , Neuronas/metabolismo , Receptor de Serotonina 5-HT2C/inmunología , Receptor de Serotonina 5-HT2C/metabolismo , Animales , Anticuerpos/inmunología , Células CHO , Cricetinae , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2C/genética , Reproducibilidad de los Resultados , Área Tegmental Ventral/citologíaRESUMEN
17beta-estradiol (E2) regulates neuronal activity via genomic and rapid, non-genomic mechanisms. The rat serotonergic neuronal cell line (RN46A) was used to investigate the rapid effects of E2 on serotonin (5-HT) reuptake and on potential intracellular signaling pathways. RN46A cells express the serotonin transporter (SERT) and estrogen receptor (ER)beta, but not ERalpha. Fifteen minute E2 treatment (10(-9)M) decreased 5-HT uptake. Intracellular cAMP levels were not increased by 15 min E2 treatment; however, E2 caused an increase in intracellular Ca2+ levels, with a maximum response within the first minute. The response was E2 specific, since other steroids (17alpha-estradiol, testosterone, and progesterone) had no effect. The ER antagonist ICI 182,780 blocked the rapid E2 effects on intracellular Ca2+ levels as did the selective ER modulator tamoxifen. In summary, changes in intracellular Ca2+ levels caused by E2 and mediated through ERbeta may be responsible for observed rapid effects of E2 on SERT activity.
Asunto(s)
Estradiol/análogos & derivados , Estrógenos/farmacología , Neuronas/metabolismo , Serotonina/metabolismo , Transducción de Señal , Andrógenos/farmacología , Animales , Calcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Estradiol/farmacología , Antagonistas de Estrógenos/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/metabolismo , Fulvestrant , Regulación de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Progesterona/farmacología , ARN Mensajero , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Tamoxifeno/farmacología , Testosterona/farmacologíaRESUMEN
We developed fixed-cell multi-well plate immunoassays that increase the throughput and ease of quantification for questions formerly assessed by immunoblot scanning. The assays make use of the now abundant antibodies designed to recognize receptor subtypes and posttranslationally modified signaling proteins. By optimizing permeabilization and fixation conditions, mainly based on specific cell types, the assay can be adapted to the study of many different antigens of importance to hormonal and neurotransmitter signaling scenarios.
Asunto(s)
Inmunoensayo/métodos , Sistema de Señalización de MAP Quinasas , Receptores de Estrógenos/análisis , Animales , Línea Celular , Células Cultivadas , Activación Enzimática , Pruebas de Enzimas/métodos , HumanosRESUMEN
The dopamine mesocorticoaccumbens pathway which originates in the ventral tegmental area (VTA) and projects to the nucleus accumbens and prefrontal cortex is a circuit important in mediating the actions of psychostimulants. The function of this circuit is modulated by the actions of serotonin (5-HT) at 5-HT(2A) receptors (5-HT(2A)R) localized to the VTA. In the present study, we tested the hypothesis that virally mediated overexpression of 5-HT(2A)R in the VTA would increase cocaine-evoked locomotor activity in the absence of alterations in basal locomotor activity. A plasmid containing the gene for the 5-HT(2A)R linked to a synthetic marker peptide (Flag) was created and the construct was packaged in an adeno-associated virus vector (rAAV-5-HT(2A)R-Flag). This viral vector (2 µl; 10(9-10) transducing units/ml) was unilaterally infused into the VTA of male rats, while control animals received an intra-VTA infusion of Ringer's solution. Virus-pretreated rats exhibited normal spontaneous locomotor activity measured in a modified open-field apparatus at 7, 14, and 21 days following infusion. After an injection of cocaine (15 mg/kg, ip), both horizontal hyperactivity and rearing were significantly enhanced in virus-treated rats (p < 0.05). Immunohistochemical analysis confirmed expression of Flag and overexpression of the 5-HT(2A)R protein. These data indicate that the vulnerability of adult male rats to hyperactivity induced by cocaine is enhanced following increased levels of expression of the 5-HT(2A)R in the VTA and suggest that the 5-HT(2A)R receptor in the VTA plays a role in regulation of responsiveness to cocaine.
RESUMEN
Allosteric modulators of the serotonin (5-HT) 5-HT(2C) receptor (5-HT(2C)R) present a unique drug design strategy to augment the response to endogenous 5-HT in a site- and event-specific manner with great potential as novel central nervous system probes and therapeutics. To date, PNU-69176E is the only reported selective positive allosteric modulator for the 5-HT(2C)R. For the first time, an optimized synthetic route to readily access PNU-69176E (1) and its diastereomer 2 has been established in moderate to good overall yields over 10 steps starting from commercially available picolinic acid. This synthetic approach not only enables a feasible preparation of a sufficient amount of 1 for use as a reference compound for secondary pharmacological studies, but also provides an efficient synthesis of key intermediates to develop novel and simplified 5-HT(2C)R allosteric modulators. Compound 1 and its diastereomer 2 were functionally characterized in Chinese hamster ovary (CHO) cells stably transfected with the 5-HT(2C)R using an intracellular calcium (Ca(i) (2+)) release assay. Compound 1 demonstrated efficacy and potency as an allosteric modulator for the 5-HT(2C)R with no intrinsic agonist activity. Compound 1 did not alter 5-HT-evoked Ca(i) (2+) in CHO cells stably transfected with the highly homologous 5-HT(2A)R. In contrast, the diastereomer 2 did not alter 5-HT-evoked Ca(i) (2+) release in 5-HT(2A)R-CHO or 5-HT(2C)R-CHO cells or exhibit intrinsic agonist activity.
Asunto(s)
Galactósidos/síntesis química , Galactósidos/farmacología , Piperidinas/síntesis química , Piperidinas/farmacología , Receptor de Serotonina 5-HT2C/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Células CHO , Calcio/metabolismo , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos/métodos , Galactósidos/química , Humanos , Piperidinas/química , Receptor de Serotonina 5-HT2C/fisiología , EstereoisomerismoRESUMEN
It is now well accepted that at least some serotonin receptors exist in dimeric and oligmeric forms. The linking of receptor ligands has been shown to have potential in the development of selective agonists and antagonists for traditionally refractive receptors. Here we report the development of a dimeric version of the known 5-HT(2A)R antagonist, M-100907. Derivatives of M-100907 were synthesized to determine an appropriate site for the linker connection. Then, homodimers with polyether linkers of different lengths were functionally tested in a bioassay to determine the optimal linker length. Attachment at the catechol of M-100907 with linkers between 12 and 18 atoms in length proved to be optimal.
RESUMEN
The 5-HT(2C)R receptor (5-HT(2C)R) exerts tonic and phasic inhibitory influence over brain circuitry, and dysregulation of this influence contributes to the neurochemical underpinnings in the etiology of a variety of neuropsychiatric disorders including addiction, depression, and schizophrenia. A strategically important regulator of the 5-HT(2C)R function and protein diversity is mRNA editing, a type of posttranscriptional modification that alters codon identity and thus the translation of distinct, though closely related, isoforms of 5-HT(2C)R from a single, original transcript. The 5-HT(2C)R mRNA can be edited at five closely spaced sites, altering the identity of up to three amino acids in the predicted second intracellular loop of the receptor to modulate receptor:G-protein coupling and constitutive activity. Methods to study changes in mRNA editing based upon direct DNA sequencing are both time and labor intensive. To streamline the acquisition of mRNA editing data and improve quantification, we have adapted real-time reverse transcription polymerase chain reaction (qRT-PCR) to detect and quantify mRNA editing in 5-HT(2C)R transcripts by utilizing TaqMan® probes modified with a minor groove binder (MGB). The method is very sensitive, detecting as little as 10⻹8 g (1 attogram) of standard cDNA template and can discriminate closely related 5-HT(2C)R mRNA edited isoforms. This technique expands the breadth of available quantification methods for mRNA editing and is particularly useful for the ex vivo analyses of mRNA editing of the 5-HT(2C)R by allowing the rapid collection of data on large numbers of tissue samples. In addition, the general technique can be adapted easily to investigate edited mRNA from other genes, thus facilitating the development of a broader knowledge base of the physiological role of mRNA editing.
Asunto(s)
Edición de ARN , ARN Mensajero/genética , Receptor de Serotonina 5-HT2C/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Encéfalo/metabolismo , Humanos , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN/aislamiento & purificación , ARN Mensajero/metabolismo , Receptor de Serotonina 5-HT2C/metabolismo , Sensibilidad y EspecificidadRESUMEN
The serotonin 2C receptor (5-HT(2C)R) plays a significant role in psychiatric disorders (e.g., depression) and is a target for pharmacotherapy. The 5-HT(2C)R is widely expressed in brain and spinal cord and is the only G-protein coupled receptor currently known to undergo mRNA editing, a post-transcriptional modification that results in translation of distinct, though closely related, protein isoforms. The 5-HT(2C)R RNA can be edited at five sites to alter up to three amino acids resulting in modulation of receptor:G-protein coupling and constitutive activity. To rapidly quantify changes ex vivo in individual 5-HT(2C)R isoform levels in response to treatment, we adapted quantitative (real-time) reverse transcription polymerase chain reaction (qRT-PCR) utilizing TaqMan probes modified with a minor groove binder (MGB). Probes were developed for four 5-HT(2C)R RNA isoforms and their sensitivity and specificity were validated systematically using standard templates. Relative expression of the four isoforms was measured in cDNAs from whole brain extracted from 129S6 and C57BL/6J mice. Rank order derived from this qRT-PCR analysis matched that derived from DNA sequencing. In mutant mice solely expressing either non-edited or fully edited 5-HT(2C)R transcripts, only expected transcripts were detected. These data suggest this qRT-PCR method is a precise and rapid means to detect closely related mRNA sequences ex vivo without the necessity of characterizing the entire 5-HT(2C)R profile. Implementation of this technique will expand and expedite studies of specific brain 5-HT(2C)R mRNA isoforms in response to pharmacological, behavioral and genetic manipulation, particularly in ex vivo studies which require rapid collection of data on large numbers of samples.
Asunto(s)
Encéfalo/metabolismo , Edición de ARN/genética , ARN Mensajero/metabolismo , Receptor de Serotonina 5-HT2C/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Empalme Alternativo/genética , Animales , Secuencia de Bases/genética , Perfilación de la Expresión Génica/métodos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Isoformas de Proteínas/genética , Procesamiento Postranscripcional del ARN/genética , Sensibilidad y Especificidad , Serotonina/metabolismo , Factores de TiempoRESUMEN
AIM: The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT(1A), 5-HT(1B), 5-HT(2A), and 5-HT(2C)) and as cAMP and intracellular Ca(2+) are modulated by different 5-HT receptors, we studied both pathways. METHODS: 5-HT uptake was determined as imipramine-inhibitable uptake of [(3)H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca(2+) changes were determined using the ratiometric method of intracellular Ca(2+) imaging. RESULTS: For uptake experiments, cells were kept for 30 min either with or without 1 microM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [(3)H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca(2+) levels either; however, adding 1 microM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca(2+) levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca(2+) levels. CONCLUSIONS: We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca(2+) levels. This suggests that 5-HT may utilize intracellular Ca(2+) to regulate 5-HT uptake.