RESUMEN
A minority of individuals experiencing traumatic events develop anxiety disorders. The reason for the lack of correspondence between the prevalence of exposure to psychological trauma and the development of anxiety is unknown. Extracellular proteolysis contributes to fear-associated responses by facilitating neuronal plasticity at the neuron-matrix interface. Here we show in mice that the serine protease neuropsin is critical for stress-related plasticity in the amygdala by regulating the dynamics of the EphB2-NMDA-receptor interaction, the expression of Fkbp5 and anxiety-like behaviour. Stress results in neuropsin-dependent cleavage of EphB2 in the amygdala causing dissociation of EphB2 from the NR1 subunit of the NMDA receptor and promoting membrane turnover of EphB2 receptors. Dynamic EphB2-NR1 interaction enhances NMDA receptor current, induces Fkbp5 gene expression and enhances behavioural signatures of anxiety. On stress, neuropsin-deficient mice do not show EphB2 cleavage and its dissociation from NR1 resulting in a static EphB2-NR1 interaction, attenuated induction of the Fkbp5 gene and low anxiety. The behavioural response to stress can be restored by intra-amygdala injection of neuropsin into neuropsin-deficient mice and disrupted by the injection of either anti-EphB2 antibodies or silencing the Fkbp5 gene in the amygdala of wild-type mice. Our findings establish a novel neuronal pathway linking stress-induced proteolysis of EphB2 in the amygdala to anxiety.
Asunto(s)
Amígdala del Cerebelo/metabolismo , Ansiedad/metabolismo , Calicreínas/metabolismo , Receptor EphB2/metabolismo , Amígdala del Cerebelo/citología , Animales , Ansiedad/genética , Trastornos de Ansiedad/etiología , Trastornos de Ansiedad/genética , Trastornos de Ansiedad/metabolismo , Conductividad Eléctrica , Miedo , Regulación de la Expresión Génica , Calicreínas/deficiencia , Calicreínas/genética , Potenciación a Largo Plazo , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal , Neuronas/metabolismo , Unión Proteica , Receptor EphB2/química , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/metabolismo , Estrés Psicológico/metabolismo , Proteínas de Unión a Tacrolimus/genéticaRESUMEN
Nicotine, the major psychoactive component of cigarette smoke, modulates neuronal activity to produce Ca2+-dependent changes in gene transcription. However, the downstream targets that underlie the long-term effects of nicotine on neuronal function, and hence behaviour, remain to be elucidated. Here, we demonstrate that nicotine administration to mice upregulates levels of the type 2 ryanodine receptor (RyR2), a Ca2+-release channel present on the endoplasmic reticulum, in a number of brain areas associated with cognition and addiction, notably the cortex and ventral midbrain. Nicotine-mediated RyR2 upregulation was driven by CREB, and caused a long-lasting reinforcement of Ca2+ signalling via the process of Ca2+-induced Ca2+ release. RyR2 upregulation was itself required for long-term phosphorylation of CREB in a positive-feedback signalling loop. We further demonstrate that inhibition of RyR-activation in vivo abolishes sensitization to nicotine-induced habituated locomotion, a well-characterised model for onset of drug dependence. Our findings, therefore, indicate that gene-dependent reprogramming of Ca2+ signalling is involved in nicotine-induced behavioural changes.
Asunto(s)
Estimulantes Ganglionares/farmacología , Plasticidad Neuronal/efectos de los fármacos , Nicotina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/genética , Regulación hacia Arriba/efectos de los fármacos , Animales , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Corteza Cerebelosa/citología , Corteza Cerebelosa/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Estimulantes Ganglionares/administración & dosificación , Masculino , Ratones , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Red Nerviosa/efectos de los fármacos , Red Nerviosa/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nicotina/administración & dosificación , Ratas , Ratas Sprague-Dawley , Canal Liberador de Calcio Receptor de Rianodina/metabolismoRESUMEN
The p53 family member TAp73 is a transcription factor that plays a key role in many biological processes, including neuronal development. In particular, we have shown that p73 drives the expression of miR-34a, but not miR-34b and c, in mouse cortical neurons. miR-34a in turn modulates the expression of synaptic targets including synaptotagmin-1 and syntaxin-1A. Here we show that this axis is retained in mouse ES cells committed to differentiate toward a neurological phenotype. Moreover, overexpression of miR-34a alters hippocampal spinal morphology, and results in electrophysiological changes consistent with a reduction in spinal function. Therefore, the TAp73/miR-34a axis has functional relevance in primary neurons. These data reinforce a role for miR-34a in neuronal development.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , MicroARNs/metabolismo , Neuritas/fisiología , Proteínas Nucleares/metabolismo , Columna Vertebral/citología , Animales , Western Blotting , Diferenciación Celular/genética , Electrofisiología , Células Madre Embrionarias/metabolismo , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Columna Vertebral/fisiología , Sinaptotagmina I/metabolismo , Sintaxina 1/metabolismoRESUMEN
Changes in synaptic strength mediated by ionotropic glutamate N-methyl-D-asparate (NMDA) receptors is generally considered to be the molecular mechanism underlying memory and learning. NMDA receptors themselves are subject to regulation through signaling pathways that are activated by G-protein-coupled receptors (GPCRs). In this study we investigate the ability of NMDA receptors to regulate the signaling of GPCRs by focusing on the G(q/11)-coupled M(3)-muscarinic receptor expressed endogenously in mouse cerebellar granule neurons. We show that NMDA receptor activation results in the phosphorylation and desensitization of M(3)-muscarinic receptors through a mechanism dependent on NMDA-mediated calcium influx and the activity of calcium-calmodulin-dependent protein kinase II. Our study reveals a complex pattern of regulation where GPCRs (M(3)-muscarinic) and NMDA receptors can feedback on each other in a process that is likely to influence the threshold value of signaling networks involved in synaptic plasticity.
Asunto(s)
Cerebelo/metabolismo , Receptor Muscarínico M3/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Secuencia de Aminoácidos , Animales , Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Cerebelo/citología , Retroalimentación Fisiológica , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , N-Metilaspartato/farmacología , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosfatidilinositoles/metabolismo , Fosforilación , Receptor Muscarínico M3/química , Receptor Muscarínico M3/deficiencia , Receptor Muscarínico M3/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
Mitochondrial fragmentation is recognized to be an important event during the onset of apoptosis. In this current study, we have used single cell imaging to investigate the role of the mitochondrial fission protein DRP-1 on mitochondrial morphology and mitochondrial fragmentation in primary hippocampal neurons undergoing necrotic or apoptotic cell death. Treatment of neurons with 500 nM staurosporine (apoptosis) or 30 µM glutamate (l-Glu; excitotoxic necrosis) produced a fragmentation and condensation of mitochondria, which although occurred over markedly different time frames appeared broadly similar in appearance. In neurons exposed to an apoptotic stimuli, inhibiting DRP-1 activity using overexpression of the dominant negative DRP-1(K38A) slowed the rate of mitochondrial fragmentation and decreased total cell death when compared to overexpression of wild-type DRP-1. In contrast, responses to l-Glu appeared DRP-1 independent. Similarly, alterations in the fission/fusion state of the mitochondrial network did not alter mitochondrial Ca(2+) uptake or the ability of l-Glu to stimulate excitotoxic Ca(2+) overload. Finally, apoptosis-induced mitochondrial fragmentation was observed concurrent with recruitment of Bax to the mitochondrial membrane. In contrast, during glutamate excitotoxicity, Bax remained in the cytosolic compartment. We conclude that different pathways lead to the appearance of fragmented mitochondria during necrotic and apoptotic neuronal cell death.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/metabolismo , Neuronas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Muerte Celular/fisiología , Células Cultivadas , Ácido Glutámico/farmacología , Hipocampo/citología , Hipocampo/metabolismo , Mitocondrias/efectos de los fármacos , Necrosis/metabolismo , Neuronas/citología , Neurotoxinas/farmacología , Plásmidos/genética , Ratas , Estaurosporina/farmacología , Transfección , Proteína X Asociada a bcl-2/metabolismoRESUMEN
High-resolution fluorescent imaging of mitochondrial-targeted probes was used to examine the ability of mitochondria to decode complex spatial and temporal Ca2+ signals evoked in synaptically active networks of hippocampal neurons. Green-to-red photoconversion of the mitochondrial-targeted probe, mito-Kaede, demonstrated that mitochondria were present as discrete organelles 2-6 microm in length. Real-time imaging of mitochondrial-targeted ratiometric pericam (2 mtRP) visualised rapid, repetitive, transient mitochondrial Ca2+ fluxes in response to periods of synaptic activation. Mitochondrial Ca2+ fluxes within cellular compartments were dependent on the extent of synaptic recruitment, but independent of cross-talk with the endoplasmic reticulum or the presence of an interconnected mitochondrial network. Mitochondria in dendritic regions demonstrated a greater sensitivity to synaptic activation compared with somatic mitochondria. Temporal decoding of synaptic signals was rate-limited by the activity of the mitochondrial Na+/Ca2+ exchanger. Spatial regulation of mitochondrial Ca2+ uptake was determined by the magnitude of the cytosolic Ca2+ rise in each cellular compartment.
Asunto(s)
Señalización del Calcio , Hipocampo/citología , Hipocampo/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Animales , Células Cultivadas , Neuronas/citología , Ratas , Sinapsis/metabolismo , Factores de TiempoRESUMEN
Relaxation of airway smooth muscle is dependent predominantly upon elevation of cell cAMP content. Although the processes involved in elevation of cell cAMP content are reasonably well established, the mechanisms governing subsequent control of cAMP turnover are less clear. Breakdown of cAMP is solely regulated by phosphodiesterase (PDE) isoenzymes. We have previously reported that PDE4 family members are likely to be important in this process, and that expression of PDE4D variants is actively regulated at the transcriptional level. Here, we demonstrate a key role for PDE4D5 in the control of beta(2)-adrenoceptor (beta(2)AR)-stimulated cAMP activity in human airway smooth muscle cells using splice variant-specific small interfering RNA knockdown. Furthermore, we show, using an Epac (exchange protein directly activated by cAMP)-based, cAMP-sensitive fluorescent probe, that these intracellular cAMP gradients are controlled both temporally and dynamically by PDE4D5. Elevation of cAMP within the cytoplasm after beta(2)AR stimulation is rapid and shows no distinct spatial compartmentalization in these cells. These data suggest that PDE4D5, despite being a minor component of the tissue PDE pool, is the key physiological regulator of beta(2)AR-induced cAMP turnover within human airway smooth muscle.
Asunto(s)
AMP Cíclico/metabolismo , Relajación Muscular/fisiología , Miocitos del Músculo Liso/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores Adrenérgicos beta 2/biosíntesis , Tráquea/enzimología , Células Cultivadas , AMP Cíclico/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Expresión Génica , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Miocitos del Músculo Liso/citología , Hidrolasas Diéster Fosfóricas/genética , ARN Interferente Pequeño/genética , Receptores Adrenérgicos beta 2/genética , Tráquea/citología , Transcripción Genética/fisiologíaRESUMEN
There is now substantial evidence, from single-cell imaging, that complex patterns of release from Ca(2+) stores play an important role in regulating synaptic efficacy and plasticity. Moreover, the major mechanism of store release depends on the generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] through the action of phospholipase(s) C on phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], and several neurotransmitters can enhance receptor-mediated activation of this enzyme. The recent development of techniques to image real-time changes in PtdIns(4,5)P(2) hydrolysis according to generation of Ins(1,4,5)P(3) and diacylglycerol in single cells has significantly advanced our ability to investigate these signalling pathways, particularly in relation to single-cell Ca(2+) signals. This article reviews these new approaches and how they have provided novel insights into mechanisms underlying spatio-temporal Ca(2+) signals and phospholipase C activation in neurons.
Asunto(s)
Señalización del Calcio/fisiología , Inositol 1,4,5-Trifosfato/fisiología , Neuronas/fisiología , Fosfatidilinositol 4,5-Difosfato/fisiología , Transducción de Señal/fisiología , Animales , Técnicas Biosensibles/métodos , Electrofisiología , Técnica del Anticuerpo Fluorescente/métodosRESUMEN
The majority of chronic kidney disease (CKD) cases have their origin in the glomerulus, the microvascular unit of the nephron that serves as a filter tasked with forming primary urine. This selective filtration process is determined to a large extent by the functional capacity of the podocyte, a highly differentiated cell type that enwraps the outer aspect of the glomerular capillary wall. In this short review, we describe the biology of the podocyte, its central role in the etiology of various glomerulopathies and highlight current and future opportunities to exploit the unique properties of this cell type for developing kidney-specific therapeutics.
Asunto(s)
Glomérulos Renales/patología , Terapia Molecular Dirigida , Podocitos/metabolismo , Animales , Diseño de Fármacos , Humanos , Insuficiencia Renal Crónica/fisiopatologíaRESUMEN
Activation of sphingosine kinase (SPHK), thereby increasing cellular levels of sphingosine 1-phosphate (S1P), may be involved in a variety of intracellular responses including Ca(2+) signaling. This study uses mammalian SPHK1a, tagged with enhanced green fluorescent protein (eGFP), to examine whether translocation of this enzyme is linked with Ca(2+)-mobilizing responses. Real-time confocal imaging of SPHK1a-eGFP in human SH-SY5Y neuroblastoma cells visualized a relocation of the enzyme from the cytosol to the plasma membrane in response to Ca(2+)-mobilizing stimuli (muscarinic M(3)- or lysophosphatidic acid receptor activation, and thapsigargin-mediated store release). This redistribution was preceded by a transient increase in cytosolic SPHK1a-eGFP levels due to liberation of SPHK from localized higher intensity regions. Translocation was dependent on Ca(2+) mobilization from intracellular stores, and was prevented by pretreatment with the Ca(2+)/calmodulin inhibitor W-7, but not W-5 or KN-62. In functional studies, pretreatment with W-7 lowered basal and M(3)-receptor-mediated cellular S1P production. However, this pretreatment did not alter agonist-mediated Ca(2+) responses, and SPHK1a-eGFP activity itself appeared insensitive to Ca(2+)/calmodulin and W-7. These data suggest a role for Ca(2+)/calmodulin in controlling the subcellular distribution but not the activity of SPHK1a.
Asunto(s)
Señalización del Calcio/fisiología , Calcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/enzimología , Células Eucariotas/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transporte de Proteínas/fisiología , Receptores Acoplados a Proteínas G , Relojes Biológicos/efectos de los fármacos , Relojes Biológicos/fisiología , Señalización del Calcio/efectos de los fármacos , Calmodulina/antagonistas & inhibidores , Membrana Celular/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Inhibidores Enzimáticos/farmacología , Células Eucariotas/efectos de los fármacos , Humanos , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Receptor Muscarínico M3 , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Receptores del Ácido Lisofosfatídico , Receptores Muscarínicos/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Proteínas Recombinantes de Fusión , Esfingosina/biosíntesis , Células Tumorales CultivadasRESUMEN
Previous studies have demonstrated that microribonucleic acids (miRs) are key regulators of protein expression in the brain and modulate dendritic spine morphology and synaptic activity. To identify novel miRs involved in neuronal plasticity, we exposed adult mice to chronic treatments with nicotine, cocaine, or amphetamine, which are psychoactive drugs that induce well-documented neuroadaptations. We observed brain region- and drug-specific changes in miR expression levels and identified miR-29a/b as regulators of synaptic morphology. In vitro imaging experiments indicated that miR-29a/b reduce mushroom-shaped dendritic spines on hippocampal neurons with a concomitant increase in filopodial-like outgrowths, suggesting an effect on synapse formation via actin cytoskeleton remodeling. We identified Arpc3, a component of the ARP2/3 actin nucleation complex, as a bona fide target for down-regulation by miR-29a/b. This work provides evidence that targeting of Arpc3 by miR-29a/b fine tunes structural plasticity by regulating actin network branching in mature and developing spines.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Espinas Dendríticas/metabolismo , MicroARNs/genética , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/genética , Actinas/metabolismo , Animales , Células Cultivadas , Espinas Dendríticas/genética , Regulación hacia Abajo , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Neuronas/metabolismo , TransfecciónRESUMEN
Inhibition of pro-survival Bcl-2 family proteins by BH3-only proteins is a key initial step leading to apoptotic cell death. In neurons, investigating cell death pathways is often hampered by the multi-factorial nature of the stress stimuli employed. Here we investigate the action of ABT-737, a small molecule inhibitor which specifically targets the BH3-protein binding domain of pro-survival Bcl-2, Bcl-X(L) and Bcl-w. ABT-737 produced a time- and concentration-dependent neuronal cell death which displayed the classical hallmarks of apoptosis. Cell death was maximal by around 4 h ABT-737 treatment, and the effect of ABT-737 could be delayed by the broad spectrum caspase inhibitor zVADfmk. Examining, using real-time confocal microscopy, the molecular basis for the onset of response demonstrated recruitment of pro-apoptotic Bax to specific mitochondrial foci, followed by mitochondrial fragmentation. Treatment of neurons with ABT-737 also produced cleavage of Bid, a BH3-only protein known to be a caspase substrate. Interestingly, cleaved Bid translocated to mitochondria but did not colocalise with Bax foci. zVADfmk inhibited Bid cleavage and slowed the rate of fragmentation, suggesting a role for cleaved Bid in the amplification of the apoptotic response. siRNA-mediated knockdown of Bax significantly inhibited ABT-737 induced cell death, whereas knockdown of the BH3-only proteins Bid or Bim had no effect. ABT-737 therefore appears to be a useful tool with which to examine neuronal apoptotic pathways. Our data suggests that caspase-dependent cleavage of Bid may be a downstream amplification event which enhances the rate of mitochondrial fragmentation.
Asunto(s)
Apoptosis/efectos de los fármacos , Compuestos de Bifenilo/farmacología , Mitocondrias/efectos de los fármacos , Neuronas/efectos de los fármacos , Nitrofenoles/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Proteína bcl-X/antagonistas & inhibidores , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cerebelo/citología , Hipocampo/citología , Mitocondrias/ultraestructura , Neuronas/ultraestructura , Piperazinas/farmacología , Unión Proteica , Estructura Terciaria de Proteína , RatasRESUMEN
From 1998-2002 twenty-five deer self-treatment devices (4-Posters), using 2% amitraz, were operated at three locations in Maryland to determine their effectiveness in controlling blacklegged ticks, Ixodes scapularis Say, and lone star ticks, Amblyomma americanum (L.). Each treatment site was approximately 518 ha and paired with a similar site lacking 4-Posters. Locations varied in deer density, tick abundance, and land use. Flagging for host-seeking ticks showed declines in tick populations at all treatment sites compared to control sites by the third year. By 2002, control of I. scapularis nymphs attributable to the 4-Poster intervention at the three sites was 69.0%, 75.8%, and 80%. Control of A. americanum nymphs at the two sites where they occurred was 99.5% and 95.3%. In 2003, the first posttreatment year, control of I. scapularis remained around 2001-2002 levels, but by 2004, an upward trend in nymphal numbers was detectable. Populations of A. americanum showed no increase posttreatment. These results demonstrate that control of these tick species is locally possible with 4-Poster intervention.
Asunto(s)
Acaricidas/administración & dosificación , Ciervos/parasitología , Control de Ácaros y Garrapatas/métodos , Infestaciones por Garrapatas/veterinaria , Alimentación Animal , Animales , Vectores Arácnidos , Humanos , Insecticidas/administración & dosificación , Modelos Lineales , Enfermedad de Lyme/prevención & control , Maryland , Densidad de Población , Control de Ácaros y Garrapatas/estadística & datos numéricos , Infestaciones por Garrapatas/prevención & control , Garrapatas/crecimiento & desarrollo , Toluidinas/administración & dosificación , Zea maysRESUMEN
Imatinib mesylate (IM), a potent inhibitor of the BCR/ABL tyrosine kinase, has become standard first-line therapy for patients with chronic myeloid leukemia (CML), but the frequency of resistance increases in advancing stages of disease. Elimination of BCR/ABL-dependent intracellular signals triggers apoptosis, but it is unclear whether this activates additional cell survival and/or death pathways. We have shown here that IM induces autophagy in CML blast crisis cell lines, CML primary cells, and p210BCR/ABL-expressing myeloid precursor cells. IM-induced autophagy did not involve c-Abl or Bcl-2 activity but was associated with ER stress and was suppressed by depletion of intracellular Ca2+, suggesting it is mechanistically nonoverlapping with IM-induced apoptosis. We further demonstrated that suppression of autophagy using either pharmacological inhibitors or RNA interference of essential autophagy genes enhanced cell death induced by IM in cell lines and primary CML cells. Critically, the combination of a tyrosine kinase inhibitor (TKI), i.e., IM, nilotinib, or dasatinib, with inhibitors of autophagy resulted in near complete elimination of phenotypically and functionally defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKIs in the treatment of CML.
Asunto(s)
Autofagia/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Células Madre Neoplásicas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Autofagia/fisiología , Benzamidas , Calcio/metabolismo , Muerte Celular/fisiología , Línea Celular Tumoral , Cloroquina/farmacología , Cloroquina/uso terapéutico , Dasatinib , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Proteínas de Fusión bcr-abl/genética , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Macrólidos/farmacología , Macrólidos/uso terapéutico , Ratones , Ratones Endogámicos C3H , Proteínas Asociadas a Microtúbulos/metabolismo , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Piperazinas/farmacología , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Interferencia de ARN , Tiazoles/farmacología , Tiazoles/uso terapéutico , Factor de Transcripción CHOP/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The metabotropic glutamate (mGlu) receptors mGlu1 and mGlu5 mediate distinct inositol 1,4,5-trisphosphate (IP(3)) and Ca(2+) signaling patterns, governed in part by differential mechanisms of feedback regulation after activation. Single cell imaging has shown that mGlu1 receptors initiate sustained elevations in IP(3) and Ca(2+), which are sensitive to agonist concentration. In contrast, mGlu5 receptors are subject to cyclical PKC-dependent uncoupling and consequently mediate coincident IP(3) and Ca(2+) oscillations that are largely independent of agonist concentration. In this study, we investigated the contribution of G(q/11)alpha protein expression levels in shaping mGlu1/5 receptor-mediated IP(3) and Ca(2+) signals, using RNA interference (RNAi). RNAi-mediated knockdown of G(q/11)alpha almost abolished the single-cell increase in IP(3) caused by mGlu1 and mGlu5 receptor activation. For the mGlu1 receptor, this unmasked baseline Ca(2+) oscillations that persisted even at maximal agonist concentrations. mGlu5 receptor-activated Ca(2+) oscillations were still observed but were only initiated at high agonist concentrations. Recombinant overexpression of G(q)alpha enhanced IP(3) signals after mGlu1 and mGlu5 receptor activation. It is noteworthy that although mGlu5 receptor-mediated IP(3) and Ca(2+) oscillations in control cells were largely insensitive to agonist concentration, increasing G(q)alpha expression converted these oscillatory signatures to sustained plateau responses in a high proportion of cells. In addition to modulating temporal Ca(2+) signals, up- or down-regulation of G(q/11)alpha expression alters the threshold for the concentration of glutamate at which a measurable Ca(2+) signal could be detected. These experiments indicate that altering G(q/11)alpha expression levels differentially affects spatiotemporal aspects of IP(3) and Ca(2+) signaling mediated by the mGlu1 and mGlu5 receptors.
Asunto(s)
Señalización del Calcio/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Glutamato Metabotrópico/fisiología , Transducción de Señal/fisiología , Animales , Secuencia de Bases , Células CHO , Cricetinae , Cartilla de ADN , Humanos , Interferencia de ARN , Receptor del Glutamato Metabotropico 5RESUMEN
The magnitude and temporal nature of intracellular signaling cascades can now be visualized directly in single cells by the use of protein domains tagged with enhanced green fluorescent protein (eGFP). In this study, signaling downstream of G protein-coupled receptor-mediated phospholipase C (PLC) activation has been investigated in a cell line coexpressing recombinant M(3) muscarinic acetylcholine and alpha(1B) -adrenergic receptors. Confocal measurements of changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)), using the pleckstrin homology domain of PLCdelta1 tagged to eGFP (eGFP-PH(PLCdelta)), and 1,2-diacylglycerol (DAG), using the C1 domain of protein kinase Cgamma (PKCgamma) (eGFP-C1(2)-PKCgamma), demonstrated clear translocation responses to methacholine and noradrenaline. Single cell EC(50) values calculated for each agonist indicated that responses to downstream signaling targets (Ca(2+) mobilization and PKC activation) were approximately 10-fold lower compared with respective Ins(1,4,5)P(3) and DAG EC(50) values. Examining the temporal profile of second messenger responses to sub-EC(50) concentrations of noradrenaline revealed oscillatory Ins(1,4,5)P(3), DAG, and Ca(2+) responses. Oscillatory recruitments of conventional (PKCbetaII) and novel (PKCepsilon) PKC isoenzymes were also observed which were synchronous with the Ca(2+) response measured simultaneously in the same cell. However, oscillatory PKC activity (as determined by translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate protein) required oscillatory DAG production. We suggest a model that uses regenerative Ca(2+) release via Ins(1,4,5)P(3) receptors to initiate oscillatory second messenger production through a positive feedback effect on PLC. By acting on various components of the PLC signaling pathway the frequency-encoded Ca(2+) response is able to maintain signal specificity at a level downstream of PKC activation.
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Calcio/metabolismo , Diglicéridos/biosíntesis , Inositol 1,4,5-Trifosfato/biosíntesis , Microscopía Confocal/métodos , Proteína Quinasa C/metabolismo , Acetilcolina/metabolismo , Animales , Técnicas Biosensibles , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Fosfatos de Inositol/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Cloruro de Metacolina/química , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Oscilometría , Fosfolipasa D/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Transducción de Señal , Factores de Tiempo , TransfecciónRESUMEN
In brain ischemia, gating of postsynaptic glutamate receptors and other membrane channels triggers intracellular Ca2+ overload and cell death. In excitotoxic settings, the initial Ca2+ influx through glutamate receptors is followed by a second uncontrolled Ca2+ increase that leads to neuronal demise. Here we report that the major plasma membrane Ca2+ extruding system, the Na+/Ca2+ exchanger (NCX), is cleaved during brain ischemia and in neurons undergoing excitotoxicity. Inhibition of Ca2+-activated proteases (calpains) by overexpressing their endogenous inhibitor protein, calpastatin or the expression of an NCX isoform not cleaved by calpains, prevented Ca2+ overload and rescued neurons from excitotoxic death. Conversely, down-regulation of NCX by siRNA compromised neuronal Ca2+ handling, transforming the Ca2+ transient elicited by non-excitotoxic glutamate concentrations into a lethal Ca2+overload. Thus, proteolytic inactivation of NCX-driven neuronal Ca2+ extrusion is responsible for the delayed excitotoxic Ca2+ deregulation and neuronal death.
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Isquemia Encefálica/fisiopatología , Membrana Celular/fisiología , Proteínas de Transporte de Membrana/metabolismo , Neuronas/fisiología , Receptores de Glutamato/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/farmacología , Calpaína/antagonistas & inhibidores , Calpaína/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Membrana Celular/efectos de los fármacos , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Datos de Secuencia Molecular , Neuronas/enzimología , Ratas , Ratas WistarRESUMEN
Using single cell Ca(2+) imaging and whole cell current clamp recordings, this study aimed to identify the signal transduction mechanisms involved in mACh receptor-mediated, enhanced synaptic signaling in primary cultures of hippocampal neurons. Activation of M(1) mACh receptors produced a 2.48 +/- 0.26-fold enhancement of Ca(2+) transients arising from spontaneous synaptic activity in hippocampal neurons. Combined imaging of spontaneous Ca(2+) signals with inositol 1,4,5-trisphosphate (IP(3)) production in single neurons demonstrated that the methacholine (MCh)-mediated enhancement required activated G(q/11)alpha subunits and phospholipase C activity but did not require measurable increases in IP(3). Electrophysiological studies demonstrated that MCh treatment depolarized neurons from -64 +/- 3 to -45 +/- 3 mV and increased action potential generation. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) enhanced neuronal excitability and prolonged the action of MCh. These studies suggest that, in addition to producing the second messengers IP(3) and diacylglycerol, mACh receptor activation may directly utilize PIP(2) hydrolysis to regulate neuronal excitability.
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Señalización del Calcio/fisiología , Hipocampo/citología , Neuronas/fisiología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Receptores Muscarínicos/fisiología , Animales , Calcio/metabolismo , Canales de Calcio/fisiología , Membrana Celular/metabolismo , Células Cultivadas , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Glutamina/fisiología , Potenciales de la Membrana/fisiología , Neuronas/citología , Ratas , Ratas Endogámicas , Receptores de Glutamato Metabotrópico/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/metabolismoRESUMEN
Inositol 1,4,5-trisphosphate (InsP(3)) production in single cerebellar granule neurons (CGNs) grown in culture was measured using the PH domain of phospholipase C delta1 tagged with enhanced green fluorescent protein (eGFP-PH(PLCdelta1)). These measurements were correlated with changes in intracellular free Ca2+ determined by single cell imaging. In control CGNs, intracellular Ca2+ stores appeared replete. However, the refilling state of these stores appeared dependent on the fluorophore used to measure Ca2+-release. Thus, methacholine (MCH), acting via muscarinic acetylcholine-receptors (mAchRs), mobilised intracellular Ca2+ in cells loaded with fluo-3 and fura-4f, but not fura-2. Confocal measurements of single CGNs expressing eGFP-PH(PLCdelta1) demonstrated that MCH stimulated a robust peak increase in InsP(3), which was followed by a sustained plateau phase of InsP(3) production. In contrast, glutamate-induced InsP(3) signals were weak or not detectable. MCH-stimulated InsP(3) production was reduced by chelation of intracellular Ca2+ with BAPTA, and emptying of intracellular stores with thapsigargin, indicated a positive feedback effect of Ca2+ mobilisation onto PLC activity. In CGNs, NMDA- and KCl-mediated Ca2+-entry significantly enhanced MCH-induced InsP(3) production. Furthermore, mAchR-mediated PLC activation appeared sensitive to the full dynamic range of intracellular Ca2+ increases stimulated by 100 microm NMDA. This dynamic regulation was also observed at the level of PKC activation indicated by an enhanced translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate (MARCKS) protein in cells stimulated with MCH. Thus, NMDA-mediated Ca2+ influx and PLC activation may represent a coincident-detection system whereby ionotropic and metabotropic signals combine to stimulate InsP(3) production and PKC-mediated phosphorylation events in CGNs.
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Calcio/metabolismo , Inositol 1,4,5-Trifosfato/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana , Neuronas/metabolismo , Proteína Quinasa C/metabolismo , Receptores Muscarínicos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Células Cultivadas , Cerebelo/citología , Activación Enzimática/fisiología , Retroalimentación Fisiológica/efectos de los fármacos , Colorantes Fluorescentes , Ácido Glutámico/farmacología , Proteínas Fluorescentes Verdes , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminiscentes/genética , Agonistas Muscarínicos/farmacología , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada , Neuronas/citología , Neuronas/efectos de los fármacos , Fosfolipasa C delta , Proteínas/genética , Ratas , Ratas Endogámicas , Receptores Muscarínicos/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , Fosfolipasas de Tipo C/genéticaRESUMEN
The dynamics of inositol 1,4,5-trisphosphate (Ins (1,4,5)P3) production during periods of G-protein-coupled receptor-mediated Ca2+ oscillations have been investigated using the pleckstrin homology (PH) domain of phospholipase C (PLC) delta1 tagged with enhanced green fluorescent protein (eGFP-PHPLCdelta1). Activation of noradrenergic alpha1B and muscarinic M3 receptors recombinantly expressed in the same Chinese hamster ovary cell indicates that Ca2+ responses to these G-protein-coupled receptors are stimulus strength-dependent. Thus, activation of alpha1B receptors produced transient base-line Ca2+ oscillations, sinusoidal Ca2+ oscillations, and then a steady-state plateau level of Ca2+ as the level of agonist stimulation increased. Activation of M3 receptors, which have a higher coupling efficiency than alpha1B receptors, produced a sustained increase in intracellular Ca2+ even at low levels of agonist stimulation. Confocal imaging of eGFP-PHPLCdelta1 visualized periodic increases in Ins(1,4,5)P3 production underlying the base-line Ca2+ oscillations. Ins(1,4,5)P3 oscillations were blocked by thapsigargin but not by protein kinase C down-regulation. The net effect of increasing intracellular Ca2+ was stimulatory to Ins(1,4,5)P3 production, and dual imaging experiments indicated that receptor-mediated Ins(1,4,5)P3 production was sensitive to changes in intracellular Ca2+ between basal and approximately 200 nM. Together, these data suggest that alpha1B receptor-mediated Ins(1,4,5)P3 oscillations result from a positive feedback effect of Ca2+ onto phospholipase C. The mechanisms underlying alpha1B receptor-mediated Ca2+ responses are therefore different from those for the metabotropic glutamate receptor 5a, where Ins(1,4,5)P3 oscillations are the primary driving force for oscillatory Ca2+ responses (Nash, M. S., Young, K. W., Challiss, R. A. J., and Nahorski, S. R. (2001) Nature 413, 381-382). For alpha1B receptors the Ca2+-dependent Ins(1,4,5)P3 production may serve to augment the existing regenerative Ca2+-induced Ca2+-release process; however, the sensitivity to Ca2+ feedback is such that only transient base-line Ca2+ spikes may be capable of causing Ins(1,4,5)P3 oscillations.