RESUMEN
Serotonergic 5-HT(1A) receptor signaling leading to nuclear factor-kappaB (NF-kappaB) activation appears to be critical for cell survival. Adenylyl cyclase and protein kinase A (AC/PKA) are effectors of the 5-HT(1A) receptor that are inhibited by Galpha(i) subunits. Conversely, Gbetagamma(i) subunits downstream from the 5-HT(1A) receptor participate in the activation of extracellular signal-regulated kinases (ERK1/2), phosphatidylinositol 3-kinase (PI3K), Akt, and NF-kappaB. To model the contribution of pro- and antiapoptotic signaling cascades downstream of activated 5-HT(1A) receptor in cell survival, Chinese hamster ovarian (CHO) cells were employed that exogenously overexpress 5-HT(1A) receptors. Stimulation with the 5-HT(1A) receptor agonist 8-OH-DPAT and pharmacological agonists of AC induced PKA and protein phosphatase 2A (PP2A) activity, which in turn inhibited: Akt activity, IkappaBalpha degradation, nuclear translocation of NF-kappaB, and expression of X-linked inhibitor of apoptosis protein (XIAP/BIRC4). Pharmacological inhibition of PP2A with calyculin A potentiated Akt activity while attenuating ERK1/2 signaling via increased inhibitory phosphorylation of Raf (pSer259). In contrast, increased cAMP levels enhanced Bax translocation to the mitochondria, resulting in the release of cytochrome c, caspase-3 activation, and apoptosis induction. Our data suggest a central role of cAMP/PKA-dependent PP2A in shifting the homeostasis of intracellular signaling downstream of activated 5-HT(1A) receptor toward cell death in biological systems linked to neuropsychiatric disorders.
Asunto(s)
Apoptosis/fisiología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Fosfatasa 2/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Transducción de Señal/fisiología , Proteína X Asociada a bcl-2/metabolismo , Animales , Western Blotting , Células CHO , Supervivencia Celular , Cricetinae , Cricetulus , Activación Enzimática/fisiología , HumanosRESUMEN
Antagonist 5-HT(1A) PET ligands are available, but an agonist ligand would give more information about signal transduction capacity. Synthesis and in vivo evaluation of [O-methyl-(11)C]2-{4-[4-(7-methoxynaphthalen-1-yl)piperazin-1-yl]butyl}-4-methyl-2H-[1,2,4]triazine-3,5-dione (10), a potential high affinity (K(i) = 1.36 nM) 5-HT(1A) agonist PET tracer is described. Piperazine 10 is a 5-HT(1A) agonist with an EC(50) comparable to serotonin, based on cAMP formation and GTP(gamma)S binding assays. Radiosynthesis of [(11)C]10 has been achieved by reacting 2-{4-[4-(7-hydroxynaphthalen-1-yl)piperazin-1-yl]butyl}-4-methyl-2H-[1,2,4]triazine-3,5-dione (9) and [(11)C]CH(3)OTf in 25 +/- 5% (n = 15) yield at the end of synthesis (EOS). The chemical and radiochemical purities of [(11)C]10 were >99% with a specific activity 1500 +/- 300 Ci/mmol (n =15). PET studies in anesthetized baboon demonstrate [(11)C]10 specific binding in brain regions rich in 5-HT(1A) receptors. Binding of [(11)C]10 was blocked by WAY100635 and 8-OH-DPAT. The regional brain volumes of distribution (V(T)) of [(11)C]10 in baboon correlate with [(11)C]WAY100635 V(T) in baboons. These data provide evidence that [(11)C]10 is the first promising agonist PET tracer for the 5-HT(1A) receptors.
Asunto(s)
Piperazinas/síntesis química , Piperazinas/farmacología , Tomografía de Emisión de Positrones/métodos , Agonistas del Receptor de Serotonina 5-HT1 , Triazinas/síntesis química , Triazinas/farmacología , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Encéfalo/efectos de los fármacos , Células CHO , Cricetinae , Ligandos , Masculino , Estructura Molecular , Papio , Piperazinas/antagonistas & inhibidores , Piridinas/farmacología , Factores de Tiempo , Distribución Tisular , Triazinas/antagonistas & inhibidoresRESUMEN
To investigate the functional consequences of cross-talk between multiple effectors of serotonin (5-HT) 1A receptor, we employed transfected Chinese hamster ovary cells. Activation of 5-HT 1A receptor stimulated extracellular signal-regulated kinase (ERK)1/2, Akt and nuclear transcription factor-kappaB (NF-kappaB). Stimulation of cells with 5-HT 1A receptor agonist induced a rapid but transient ERK1/2 phosphorylation followed by increased phosphorylation of Akt. Elevated Akt activity in turn suppressed Raf activity and induced a decline in ERK activation. The activation of ERK and Akt downstream of 5-HT 1A receptor was sensitive to inhibitors of Ras, Raf and phosphatidylinositol 3-kinase (PI3K). Stimulation of 5-HT 1A receptor also resulted in activation of NF-kappaB through a decrease in inhibitor of nuclear transcription factor-kappaB. In support of the importance of 5-HT 1A receptor signaling for cell survival, inhibition of NF-kappaB facilitated caspase 3 activation and cleavage of poly (ADP-ribose) polymerase, while treatment of cells with agonist inhibited caspase 3, DNA fragmentation and cell death. Both agonist-dependent NF-kappaB activation and cell survival were decreased by Akt Inhibitor II or by overexpression of dominant-negative Akt. These findings suggest a role for 5-HT 1A receptor signaling in the Ras/Raf-dependent regulation of multiple intracellular signaling pathways that include ERK and PI3K/Akt. Of these, only PI3K/Akt and NF-kappaB activation were required for 5-HT 1A receptor-dependent cell survival, implying that the relative distribution of signals between competing transduction pathways determines the functional outcome of 5-HT 1A receptor activation.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/fisiología , FN-kappa B/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Receptor de Serotonina 5-HT1A/efectos de los fármacos , Agonistas de Receptores de Serotonina/farmacología , Transducción de Señal/fisiología , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Adenoviridae/genética , Animales , Western Blotting , Células CHO , Caspasa 3 , Caspasas/metabolismo , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Cricetinae , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Vectores Genéticos , Humanos , Toxina del Pertussis/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasas/metabolismo , Piridinas/farmacología , Azul de TripanoRESUMEN
Mice lacking dopamine D2 receptors exhibit a significantly decreased agonist-promoted forebrain neocortical D1 receptor activation that occurs without changes in D1 receptor expression levels. This raises the possibility that, in brains of D2 mutants, a substantial portion of D1 receptors are uncoupled from their G protein, a phenomenon known as receptor desensitization. To test this, we examined D1-agonist-stimulated [35S]GTPgammaS binding (in the presence and absence of protein phosphatase inhibitors) and cAMP production (in the presence and absence of pertussis toxin) in forebrain neocortical tissues of wild-type mice and D2-receptor mutants. These studies revealed a decreased agonist-stimulated G-protein activation in D2 mutants. Moreover, whereas protein phosphatase 1/2A (PP1/2A) and 2B (PP2B) inhibitors decrease [35S]GTPgammaS binding in a concentration-dependent manner in wild type, they have either no (PP2B) or only partial (PP1/2A) effects in D2 mutants. Furthermore, for D2 mutants, immunoprecipitation experiments revealed increased basal and D1-agonist-stimulated phosphorylation of D1-receptor proteins at serine residues. Finally, D1 immunoprecipitates of both wild type and D2 mutants also contain protein kinase A (PKA) and PP2B immunoreactivities. In D2 mutants, however, the catalytic activity of the immunoprecipitated PP2B is abolished. These data indicate that neocortical D1 receptors are physically linked to PKA and PP2B and that the increased phosphorylation of D1 receptors in brains of D2 mutants is due to defective dephosphorylation of the receptor rather than increased kinase-mediated phosphorylation.
Asunto(s)
Calcineurina/metabolismo , Receptores de Dopamina D1/metabolismo , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Inhibidores de la Calcineurina , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacología , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Sustancias Macromoleculares , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Toxina del Pertussis/farmacología , Fosfoserina/análisis , Fosfoserina/metabolismo , Proteína Fosfatasa 1 , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/química , Receptores de Dopamina D2/deficiencia , Receptores de Dopamina D2/genéticaRESUMEN
Positron emission tomography studies in major depression show reduced serotonin (5-HT)1A receptor antagonist-binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5-HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Measurements of [35S]GTPgammaS binding to Galphai/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5-HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Galphai/o, Galphaq/11 or Galphas proteins, or in the activity of cAMP-dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5-HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3-kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate. Finally, the activation of extracellular signal-regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist-stimulated 5-HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5-HT1A receptor transduction pathways that may have implications for cell survival.
Asunto(s)
Encéfalo/metabolismo , Proteínas Serina-Treonina Quinasas , Receptores de Serotonina/metabolismo , Sistemas de Mensajero Secundario , Transducción de Señal , Suicidio , Adenilil Ciclasas/efectos de los fármacos , Adenilil Ciclasas/metabolismo , Adulto , Anciano , Química Encefálica , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Depresión/metabolismo , Femenino , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Lóbulo Occipital/química , Lóbulo Occipital/metabolismo , Fosfohidrolasa PTEN , Toxina del Pertussis/química , Fosfatidilinositol 3-Quinasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Receptores de Serotonina 5-HT1 , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Elevation of extracellular Ca2+ (increase[Ca2+]e) stimulates the Ca2+ receptor (CaR) to induce secretion of 5-hydroxytryptamine (5-HT) from the calcium-sensing parafollicular (PF) cells. The CaR has been reported to couple to Galpha(q) with subsequent activation of protein kinase C-gamma (PKCgamma). We have identified a parallel transduction pathway in primary cultures of sheep PF cells by using a combinatorial approach in which we expressed adenoviral-encoded dominant-negative signaling proteins and performed in vitro kinase assays. The role of the CaR was established by expression of a dominant-negative CaR that eliminated calcium-induced 5-HT secretion but not secretion in response to KCl or phorbol esters. The calcium-induced secretion was inhibited by a dominant-negative p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3-K). PI3-K activity was also assayed using isoform-specific antibodies. The activity of p85/p110beta (PI3-Kbeta) immunocomplexes was elevated by increase[Ca2+]e and activated by Gbetagamma subunits. In addition, secretion of 5-HT was antagonized by the expression of a minigene encoding a peptide scavenger of Gbetagamma subunits (C-terminal fragment peptide of bovine beta-adrenergic receptor kinase). One target of PI3-K activity is phosphoinositide-dependent kinase-1 (PDK1), which in turn activated PKCzeta. Expression of a dominant-negative PKCzeta in PF cells reduced 5-HT secretion. Together, these observations establish that increase[Ca2+]e evokes 5-HT secretion from PF cells by stimulating both Galpha(q)- and Gbetagamma-signaling pathways downstream of the CaR. The betagamma cascade subsequently activates PI3-Kbeta-dependent signaling that is coupled to PDK1 and the downstream effector PKCzeta, and results in an increase in 5-HT release.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Serotonina/metabolismo , Transducción de Señal/fisiología , Glándula Tiroides/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Calcio/metabolismo , Calcio/farmacología , Proteínas de Unión al Calcio/genética , Separación Celular , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Espacio Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Expresión Génica , Genes Dominantes , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/genética , Fosfatidilinositol 3-Quinasas/genética , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ovinos , Transducción de Señal/efectos de los fármacos , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Transfección , Quinasas de Receptores Adrenérgicos betaRESUMEN
Trapping of weak bases was utilized to evaluate stimulus-induced changes in the internal pH of the secretory vesicles of chromaffin cells and enteric neurons. The internal acidity of chromaffin vesicles was increased by the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP; in vivo and in vitro) and by high K+ (in vitro); and in enteric nerve terminals by exposure to veratridine or a plasmalemmal [Ca2+]o receptor agonist (Gd3+). Stimulation-induced acidification of chromaffin vesicles was [Ca2+]o-dependent and blocked by agents that inhibit the vacuolar proton pump (vH+-ATPase) or flux through Cl- channels. Stimulation also increased the average volume of chromaffin vesicles and the proportion that displayed a clear halo around their dense cores (called active vesicles). Stimulation-induced increases in internal acidity and size were greatest in active vesicles. Stimulation of chromaffin cells in the presence of a plasma membrane marker revealed that membrane was internalized in endosomes but not in chromaffin vesicles. The stable expression of botulinum toxin E to prevent exocytosis did not affect the stimulation-induced acidification of the secretory vesicles of mouse neuroblastoma Neuro2A cells. Stimulation-induced acidification thus occurs independently of exocytosis. The quantal size of secreted catecholamines, measured by amperometry in cultured chromaffin cells, was found to be increased either by prior exposure to L-DOPA or stimulation by high K+, and decreased by inhibition of vH+-ATPase or flux through Cl- channels. These observations are consistent with the hypothesis that the content of releasable small molecules in secretory vesicles is increased when the driving force for their uptake is enhanced, either by increasing the transmembrane concentration or pH gradients.