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1.
J Neurosci ; 19(10): 3674-80, 1999 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-10233999

RESUMEN

The present study demonstrates that the regulator of G-protein-signaling protein type 4 (RGS4) is differentially regulated in the locus coeruleus (LC) and the paraventricular nucleus (PVN) of the hypothalamus by chronic stress and glucocorticoid treatments. Acute or chronic administration of corticosterone to adult rats decreased RGS4 mRNA levels in the PVN but increased these levels in the LC. Similarly, chronic unpredictable stress decreased RGS4 mRNA levels in the PVN but had a strong trend to increase these levels in the LC. Chronic stress also decreased RGS4 mRNA levels in the pituitary. The molecular mechanisms of RGS4 mRNA regulation were further investigated in vitro in the LC-like CATH.a cell line and the neuroendocrine AtT20 cell line using the synthetic corticosterone analog dexamethasone. Consistent with the findings in vivo, dexamethasone treatment caused a dose- and time-dependent decrease in RGS4 mRNA levels in AtT20 cells but a dose- and time-dependent increase in CATH.a cells. RGS4 mRNA regulation seen in these two cell lines seems to be attributable, at least in part, to opposite changes in mRNA stability. The differential regulation of RGS4 expression in the LC and in key relays of the hypothalamic-pituitary-adrenal axis could contribute to the brain's region-specific and long-term adaptations to stress.


Asunto(s)
Encéfalo/fisiología , Glucocorticoides/farmacología , Proteínas del Tejido Nervioso/fisiología , Proteínas/fisiología , Proteínas RGS , Estrés Fisiológico/fisiopatología , Animales , Células Cultivadas , Enfermedad Crónica , Corticosterona/farmacología , AMP Cíclico/fisiología , Dexametasona/farmacología , Retroalimentación , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba
2.
Brain Res Mol Brain Res ; 51(1-2): 115-22, 1997 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-9427513

RESUMEN

To investigate the receptor-channel coupling pathway, the coding region of the 5-HT1a receptor was subcloned into two plasmid vectors pSP64(polyA+) and pSP64T. Compared to the original 5-HT1a receptor construct G-21, both new constructs increased greatly the expression of functional 5-HT1a receptors in Xenopus oocytes, which developed large inward current responses to 5-HT. These responses were dose-dependent (EC50 approximately 150 nM), and could be elicited also by 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The 5-HT1a receptor mediated current had an oscillatory time course, and a reversal potential close to the equilibrium potential for Cl- (ca. -25 mV). Moreover, during and for some minutes following the application of 5-HT, these oocytes acquired the property of generating a transient inward current when their membrane was hyperpolarized. These features are characteristic of responses mediated by other receptors (e.g. muscarinic, angiotensin, serum receptors, etc.) that are known to couple to the endogenous PLC/PI second messenger pathway in Xenopus oocytes. In particular, the 5-HT1a receptor mediated current was very similar to the current induced by 5-HT-stimulation of heterogenic 5-HT2c receptors. Our results show further that the 5-HT1a receptor couples to the endogenous PLC/PI pathway much less efficiently than the 5-HT2c receptor. These results demonstrate clearly that the human 5-HT1a receptor can couple efficiently to the Xenopus oocyte endogenous PLC/PI pathway, and provide additional evidence for cell-specific signal transduction.


Asunto(s)
8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Oocitos/fisiología , Receptores de Serotonina/fisiología , Serotonina/farmacología , Fosfolipasas de Tipo C/metabolismo , Animales , Clonación Molecular , Dopamina/farmacología , Femenino , Humanos , Potenciales de la Membrana/efectos de los fármacos , Norepinefrina/farmacología , Fosfatidilinositoles/metabolismo , Biosíntesis de Proteínas , Ratas , Receptor de Serotonina 5-HT2C , Receptores de Serotonina/biosíntesis , Receptores de Serotonina 5-HT1 , Proteínas Recombinantes/biosíntesis , Sistemas de Mensajero Secundario , Agonistas de Receptores de Serotonina/farmacología , Transcripción Genética , Xenopus
3.
Brain Res Mol Brain Res ; 89(1-2): 11-9, 2001 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-11311971

RESUMEN

It is known that 5-HT receptors have significant roles in nociceptive and motor functions. We have compared the cellular localization of the mRNAs encoding serotonin 5-HT(2A,) 5-HT(2C,) 5-HT(3) receptor subtypes within different levels of the rat spinal cord and medulla. In the spinal cord, 5-HT(2C) receptor mRNA is expressed at high levels in most of the gray matter, except for lamina II. In contrast, 5-HT(2A) receptor mRNA is expressed exclusively in lamina IX. 5-HT(3) receptor mRNA has a low level and diffuse pattern of expression increasing towards the ventral horn. In both gray and white matter, there is a characteristic presence of a few highly stained cells. For each subtype, the expression pattern is similar in all four levels of the spinal cord. In the medulla, 5-HT(2C) receptor mRNA is at high levels in many nuclei including the hypoglossal nucleus, the gigantocellular reticular nucleus alpha and the parvocellular reticular nucleus alpha, the spinal nucleus of the trigeminal tract, the facial, and the dorsal medullary reticular field. Moderate to low levels of expression are seen in the spinal vestibular nucleus, the vagus, the solitary nuclei and the raphe. 5-HT(2A) receptor is expressed at high levels in some nuclei such as the hypoglossal nucleus, the intercalate nucleus, the inferior olive and the lateral reticular nucleus. Moderate to low levels of expression are seen in the facial, the medial vestibular nuclei, the nucleus ambiguous, the vagus, and the gigantocellular reticular nucleus. 5-HT(3) receptor mRNA is present at low levels in most of the nuclei examined, with a few scattered strongly labeled cells. The results show a distinct distribution of the three subtypes of receptors supporting their physiological roles and will help to understand the mechanisms of nociception and motor function.


Asunto(s)
Bulbo Raquídeo/fisiología , Receptores de Serotonina/genética , Médula Espinal/fisiología , Animales , Femenino , Expresión Génica/fisiología , Hibridación in Situ , Masculino , Bulbo Raquídeo/química , Nociceptores/fisiología , ARN Mensajero/análisis , Ratas , Ratas Wistar , Receptor de Serotonina 5-HT2A , Receptor de Serotonina 5-HT2C , Receptores de Serotonina 5-HT3 , Médula Espinal/química
4.
Proc Natl Acad Sci U S A ; 94(5): 2036-40, 1997 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-9050900

RESUMEN

Fluoxetine (Prozac) inhibited the membrane currents elicited by serotonin (5-hydroxytryptamine; 5HT) in Xenopus oocytes expressing either cloned 5HT2C receptors or 5HT receptors encoded by rat cortex mRNA. Responses of 5HT2C receptors, elicited by nM concentrations of 5HT, were rapidly and reversibly blocked by micromolar concentrations of fluoxetine. For responses elicited by 1 microM 5HT, the IC50 of fluoxetine inhibition was approximately 20 microM. In accord with the electrophysiological results, fluoxetine inhibited the binding of [3H]5HT to 5HT2C receptors expressed in HeLa cells (Ki approximately 65-97 nM), and the binding to 5HT receptors in rat cortex membranes was also inhibited but less efficiently (Ki approximately 56 microM). Our results show that fluoxetine is a competitive and reversible antagonist of 5HT2C receptors and suggest that some therapeutic effects of fluoxetine may involve blockage of 5HT receptors, in addition to its known blockage of 5HT transporters. Similar work may help to design more selective compounds for use in the treatment of brain disorders.


Asunto(s)
Fluoxetina/farmacología , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Receptores de Serotonina/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Angiotensina III/farmacología , Animales , Unión Competitiva , Encéfalo/metabolismo , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Relación Dosis-Respuesta a Droga , Expresión Génica , Células HeLa , Humanos , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/metabolismo , Oocitos , Técnicas de Placa-Clamp , Unión Proteica/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Receptor de Serotonina 5-HT2C , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Transfección/genética , Xenopus
5.
Proc Natl Acad Sci U S A ; 94(6): 2715-8, 1997 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-9122262

RESUMEN

To determine if N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a carboxyl group activating agent, can inactivate 5HT2c receptors, we have examined the effects of EEDQ on 5HT2c receptor-mediated responses to 5-hydroxytryptamine (5HT) in Xenopus oocytes, and on the binding of [3H]5HT to 5HT2c receptors in transfected HeLa cells. In oocytes expressing rat 5HT2c receptors, EEDQ inhibited the 5HT2c receptor-mediated Cl- currents; and the response did not recover more than 24 h after removal of the EEDQ. To see if this effect of EEDQ was on the receptor itself, the binding of 5HT to 5HT2c receptors was studied in transfected HeLa cells. EEDQ decreased the specific binding of [3H]5HT to 5HT2c receptors. At approximately 22 degrees C, incubating the membranes with 2 x 10(-4) M EEDQ for 1 h caused a 40% decrease in the Bmax, without changing the K(d). At 37 degrees C, the same treatment with EEDQ blocked [3H]5HT binding completely. Half-maximal inhibition occurred at 5 microM EEDQ at both temperatures, and washing for 1.5 h did not restore the binding, suggesting that the inactivation of 5HT2c receptor binding was practically irreversible. Results from both systems showed clearly that EEDQ is an irreversible antagonist of 5HT2c receptors and therefore can be used for many studies of this receptor.


Asunto(s)
Quinolinas/farmacología , Receptores de Serotonina/fisiología , Antagonistas de la Serotonina/farmacología , Animales , Corteza Cerebral/metabolismo , Femenino , Células HeLa , Humanos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Canales de Potasio/efectos de los fármacos , Ratas , Receptor de Serotonina 5-HT2C , Receptores de Serotonina/biosíntesis , Receptores de Serotonina/efectos de los fármacos , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Serotonina/metabolismo , Transfección , Xenopus laevis
6.
J Neurosci ; 17(20): 8024-37, 1997 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-9315921

RESUMEN

The recently discovered regulators of G-protein signaling (RGS) proteins potently modulate the functioning of heterotrimeric G-proteins by stimulating the GTPase activity of G-protein alpha subunits. The mRNAs for numerous subtypes of putative RGS proteins have been identified in mammalian tissues, but little is known about their expression in brain. We performed a systematic survey of the localization of mRNAs encoding nine of these RGSs, RGS3-RGS11, in brain by in situ hybridization. Striking region-specific patterns of expression were observed. Five subtypes, RGS4, RGS7, RGS8, RGS9, and RGS10 mRNAs, are densely expressed in brain, whereas the other subtypes (RGS3, RGS5, RGS6, and RGS11) are expressed at lower density and in more restricted regions. RGS4 mRNA is notable for its dense expression in neocortex, piriform cortex, caudoputamen, and ventrobasal thalamus. RGS8 mRNA is highly expressed in the cerebellar Purkinje cell layer as well as in several midbrain nuclei. RGS9 mRNA is remarkable for its nearly exclusive enrichment in striatal regions. RGS10 mRNA is densely expressed in dentate gyrus granule cells, superficial layers of neocortex, and dorsal raphe. To assess whether the expression of RGS mRNAs can be regulated, we examined the effect of an acute seizure on levels of RGS7, RGS8, and RGS10 mRNAs in hippocampus. Of the three subtypes, changes in RGS10 levels were most pronounced, decreasing by approximately 40% in a time-dependent manner in response to a single seizure. These results, which document highly specific patterns of RGS mRNA expression in brain and their ability to be regulated in a dynamic manner, support the view that RGS proteins may play an important role in determining the intensity and specificity of signaling pathways in brain as well as their adaptations to synaptic activity.


Asunto(s)
Encéfalo/metabolismo , Proteínas/metabolismo , Animales , Autorradiografía , Northern Blotting , Proteínas Activadoras de GTPasa , Masculino , Proteínas/clasificación , Proteínas/genética , ARN Complementario/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Convulsiones/metabolismo , Distribución Tisular
7.
J Biol Chem ; 276(33): 30942-7, 2001 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-11408487

RESUMEN

Inactivation of serotonin transporter (SERT) expressed in HeLa cells by [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET) occurred much more readily when Na(+) in the reaction medium was replaced with Li(+). This did not result from a protective effect of Na(+) but rather from a Li(+)-specific increase in the reactivity of Cys-109 in the first external loop of the transporter. Li(+) alone of the alkali cations caused this increase in reactivity. Replacing Na(+) with N-methyl-d-glucamine (NMDG(+)) did not reduce the affinity of cocaine for SERT, as measured by displacement of a high affinity cocaine analog, but replacement of Na(+) with Li(+) led to a 2-fold increase in the K(D) for cocaine. The addition of either cocaine or serotonin (5-HT) protected SERT against MTSET inactivation. When SERT was expressed in Xenopus oocytes, inward currents were elicited by superfusing the cell with 5-HT (in the presence of Na(+)) or by replacing Na(+) with Li(+) but not NMDG(+). MTSET treatment of oocytes in Li(+) but not in Na(+) decreased both 5-HT and Li(+) induced currents, although 5-HT-induced currents were inhibited to a greater extent. Na(+) antagonized the effects of Li(+) on both inactivation and current. These results are consistent with Li(+) inducing a conformational change that exposes Cys-109, decreases cocaine affinity, and increases the uncoupled inward current.


Asunto(s)
Proteínas Portadoras/química , Cocaína/metabolismo , Litio/farmacología , Glicoproteínas de Membrana/química , Proteínas de Transporte de Membrana , Proteínas del Tejido Nervioso , Animales , Cisteína , Glutamatos/farmacología , Células HeLa , Humanos , Potenciales de la Membrana/efectos de los fármacos , Mesilatos/farmacología , Conformación Proteica , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Sodio/farmacología , Xenopus
8.
J Neurosci ; 19(6): 2016-26, 1999 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-10066255

RESUMEN

Regulators of G-protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for alpha subunits of heterotrimeric G-proteins. Previous in situ hybridization analysis of mRNAs encoding RGS3-RGS11 revealed region-specific expression patterns in rat brain. RGS9 showed a particularly striking pattern of almost exclusive enrichment in striatum. In a parallel study, RGS9 cDNA, here referred to as RGS9-1, was cloned from retinal cDNA libraries, and the encoded protein was identified as a GAP for transducin (Galphat) in rod outer segments. In the present study we identify a novel splice variant of RGS9, RGS9-2, cloned from a mouse forebrain cDNA library, which encodes a striatal-specific isoform of the protein. RGS9-2 is 191 amino acids longer than the retinal isoform, has a unique 3' untranslated region, and is highly enriched in striatum, with much lower levels seen in other brain regions and no expression detectable in retina. Immunohistochemistry showed that RGS9-2 protein is restricted to striatal neuropil and absent in striatal terminal fields. The functional activity of RGS9-2 is supported by the finding that it, but not RGS9-1, dampens the Gi/o-coupled mu-opioid receptor response in vitro. Characterization of a bacterial artificial chromosome genomic clone of approximately 200 kb indicates that these isoforms represent alternatively spliced mRNAs from a single gene and that the RGS domain, conserved among all known RGS members, is encoded over three distinct exons. The distinct C-terminal domains of RGS9-2 and RGS9-1 presumably contribute to unique regulatory properties in the neural and retinal cells in which these proteins are selectively expressed.


Asunto(s)
Empalme Alternativo , Clonación Molecular , Cuerpo Estriado/metabolismo , Proteínas/genética , Proteínas/fisiología , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , ADN Complementario/genética , Proteínas Activadoras de GTPasa , Genoma , Isomerismo , Ratones , Datos de Secuencia Molecular , Receptores Opioides mu/fisiología , Distribución Tisular/fisiología
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