RESUMO
This work studies the deposition of aluminium-incorporated nickel coatings by brush electroplating, focusing on the electroplating setup and processing parameters. The setup was optimised in order to increase the volume of particle incorporation. The optimised design focused on increasing the plating solution flow to avoid sedimentation, and as a result the particle transport experienced a three-fold increase when compared with the traditional setup. The influence of bath load, current density and the brush material used was investigated. Both current density and brush material have a significant impact on the morphology and composition of the coatings. Higher current densities and non-abrasive brushes produce rough, particle-rich samples. Different combinations of these two parameters influence the surface characteristics differently, as illustrated in a Structure Zone Diagram. Finally, surfaces featuring crevices and peaks incorporate between 3.5 and 20 times more particles than smoother coatings. The presence of such features has been quantified using average surface roughness Ra and Abbott-Firestone curves. The combination of optimised setup and rough surface increased the particle content of the composite to 28 at.%.
RESUMO
We aimed to test the hypothesis that self-selecting fluid intake but maintaining high exogenous CHO availability (60 g/h) does not compromise half-marathon performance. 15 participants completed 3 half-marathons while drinking a 6% CHO solution to guidelines (DRINK) or a non-caloric solution in self-selected volumes when consuming 3×glucose (20 g) gels (G-GEL) or glucose-fructose (13 g glucose+7 g fructose) gels (GF-GEL) per hour. Fluid intake (DRINK: 1 557±182, G-GEL: 473±234, GF-GEL: 404±144 ml) and percent body mass loss (DRINK: - 0.8±0.9, G-GEL: - 2.0±0.6, GF-GEL: -2.3±1.1) were different (P<0.05) between conditions, though race time did not differ (DRINK: 110.6±14.4, G-GEL: 110.3±14.6, GF-GEL: 113.7±12.8 min). In G-GEL, there was a positive correlation (P<0.05) between body mass loss and race time. Plasma glucose was lower (P<0.05) in GF-GEL compared with other conditions, and total CHO oxidation (DRINK: 3.2±0.5, G-GEL: 3.0±0.4, GF-GEL: 2.6±0.4 g/min) was lower (P=0.06) in this trial. Self-selecting fluid intake but maintaining high CHO availability does not impair half-marathon performance. Additionally, consuming glucose-fructose mixtures in sub-optimal amounts reduces plasma glucose and total rates of CHO oxidation.
Assuntos
Bebidas , Carboidratos da Dieta/administração & dosagem , Resistência Física/fisiologia , Soluções para Reidratação , Corrida/fisiologia , Adulto , Glicemia/metabolismo , Índice de Massa Corporal , Carboidratos da Dieta/metabolismo , Metabolismo Energético , Feminino , Frutose/administração & dosagem , Glucose/administração & dosagem , Humanos , Masculino , Adulto JovemRESUMO
Stainless steel, polymethylmethacrylate and polytetrafluoroethylene coupons were analysed for surface topographical and adhesion force characteristics using tapping mode atomic force microscopy and force-distance microscopy techniques. The two polymer materials were surface modified by polishing with silicon carbide papers of known grade. The struvite scaling rate was determined for each coupon and related to the data gained from the surface analysis. The scaling rate correlated well with adhesion force measurements indicating that lower energy materials scale at a lower rate. The techniques outlined in the paper provide a method for the rapid screening of materials in potential scaling applications.
Assuntos
Hemostáticos/química , Compostos de Magnésio/química , Fosfatos/química , Abastecimento de Água , Precipitação Química , Corrosão , Teste de Materiais , Microscopia de Força Atômica , Polimetil Metacrilato/química , Politetrafluoretileno/química , Aço/química , EstruvitaRESUMO
Depolarization of neurons can lead to changes in gene expression that are important for such processes as synaptic plasticity, neuronal differentiation, and apoptosis. Impey and Goodman discuss some of the opposing models for how gene transcription in response to neuronal activity and elevations in intracellular calcium concentration is regulated. The pathways appear to converge on cyclic AMP response element-binding (CREB) protein, with the mitogen-activated protein kinase pathways playing an important role. The continuing debate about the involvement of calmodulin kinase IV is also described.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismoRESUMO
Cyclic AMP-response element modulator alpha (CREMalpha) is a transcription factor that is highly related to cAMP-response element-binding protein (CREB) but represses cAMP-induced gene expression from simple artificial promoters containing a cAMP-response element (CRE). CREMalpha lacks two glutamine-rich Q regions that, in CREB, are thought to be necessary for transcriptional activation. Nevertheless, protein kinase A stimulation induces CREMalpha to activate the complex native promoter in the phosphoenolpyruvate carboxykinase (PEPCK) gene. To study this phenomenon in the absence of protein kinase A stimulation, we introduced a mutation into CREMalpha to allow constitutive binding to the coactivator CREB-binding protein. This mutant, CREMalpha(DIEDML), constitutively activated the PEPCK promoter. By engineering the leucine zipper regions of CREMalpha(DIEDML) and CREB(DIEDML) to direct their patterns of dimerization, we found that only CREMalpha(DIEDML) homodimers fully activated the PEPCK promoter. By using a series of deletion and block mutants of the PEPCK promoter, we found that activation by CREMalpha(DIEDML) depended on the CRE and two CCAAT/enhancer-binding protein (C/EBP) sites. A dominant negative inhibitor of C/EBP, A-C/EBP, suppressed activation by CREMalpha(DIEDML). Furthermore, a GAL4-C/EBPalpha fusion protein and CREMalpha(DIEDML) cooperatively activated a promoter containing three GAL4 sites and the PEPCK CRE. Thus, we propose that the C/EBP sites in the PEPCK promoter allow CREMalpha to activate transcription despite its lack of Q regions.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas Repressoras , Ativação Transcricional/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Modulador de Elemento de Resposta do AMP Cíclico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Dados de Sequência Molecular , Mutação , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Células Tumorais CultivadasRESUMO
Recent evidence indicates that phosphatidylinositol 3-kinase (PI3K) is a central regulator of mitosis, apoptosis and oncogenesis. Nevertheless, the mechanisms by which PI3K regulates proliferation are not well characterized. Mitogens stimulate entry into the cell cycle by inducing the expression of immediate early genes (IEGs) that in turn trigger the expression of G(1) cyclins. Here we describe a novel PI3K- regulated transcriptional cascade that is critical for mitogen regulation of the IEG, c-fos. We show that PI3K activates gene expression by transactivating SRF-dependent transcription independently of the previously described Rho and ETS TCF pathways. PI3K-stimulated cell cycle progression requires transactivation of SRF and expression of dominant- negative PI3K blocks mitogen-stimulated cell cycle progression. Furthermore, dominant-interfering SRF mutants attenuate mitogen-stimulated cell cycle progression, but are without effect on MEK-stimulated cell cycle entry. Moreover, expression of constitutively active SRF is sufficient for cell cycle entry. Thus, we delineate a novel SRF-dependent mitogenic cascade that is critical for PI3K- and growth factor-mediated cell cycle progression.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Células 3T3 , Androstadienos/farmacologia , Animais , Western Blotting , Ciclo Celular , Divisão Celular , Separação Celular , Cromonas/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Proteínas de Ligação ao GTP/metabolismo , Genes Dominantes , Células HeLa , Humanos , Imuno-Histoquímica , Óperon Lac , Luciferases/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno , Morfolinas/farmacologia , Mutagênese Sítio-Dirigida , Fator de Crescimento Neural/farmacologia , Células PC12 , Fosforilação , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Resposta Sérica , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transfecção , Wortmanina , Proteínas Quinases p38 Ativadas por Mitógeno , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Regulation of gene transcription via the cAMP-mediated second messenger pathway has been implicated in the actions of antidepressant drugs, but studies to date have not demonstrated such an effect in vivo. To directly study the regulation of cAMP response element (CRE)-mediated gene transcription by antidepressants, transgenic mice with a CRE-LacZ reporter gene construct were administered one of three different classes of antidepressants: a norepinephrine selective reuptake inhibitor (desipramine), a serotonin selective reuptake inhibitor (fluoxetine), or a monoamine oxidase inhibitor (tranylcypromine). Chronic, but not acute, administration of these antidepressants significantly increased CRE-mediated gene transcription, as well as the phosphorylation of CRE binding protein (CREB), in several limbic brain regions thought to mediate the action of antidepressants, including the cerebral cortex, hippocampus, amygdala, and hypothalamus. These results demonstrate that chronic antidepressant treatment induces CRE-mediated gene expression in a neuroanatomically differentiated pattern and further elucidate the molecular mechanisms underlying the actions of these widely used therapeutic agents.
Assuntos
Antidepressivos/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação para Cima/efeitos dos fármacos , Animais , Antidepressivos de Segunda Geração/farmacologia , Antidepressivos Tricíclicos/farmacologia , Antipsicóticos/farmacologia , Química Encefálica/efeitos dos fármacos , Cocaína/farmacologia , Corantes , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Desipramina/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Fluoxetina/farmacologia , Haloperidol/farmacologia , Óperon Lac/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Inibidores da Monoaminoxidase/farmacologia , Fosforilação , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Tranilcipromina/farmacologia , beta-Galactosidase/biossíntese , beta-Galactosidase/genéticaRESUMO
Gene transcription is thought to be essential for memory consolidation and long-lasting changes in synaptic function. In particular, the signal transduction pathways that activate the transcription factor cAMP response element binding protein (CREB) have been implicated in the process of synaptic potentiation. To study the involvement of this pathway in neocortical plasticity within the barrel cortex, we have used a strain of mice carrying a LacZ reporter gene with six cAMP response elements (CREs) upstream of a minimal promoter. Removal of all but one facial whisker results in the expansion of the spared whisker's functional representation within somatosensory cortex. Under the same conditions of whisker deprivation, we observed a strong (eightfold compared with baseline) and highly place-specific upregulation of CRE-mediated gene transcription in layer IV of the spared whisker barrel. Reporter gene upregulation occurred rapidly after deprivation (16 hr) and was only observed under experimental conditions capable of inducing whisker response potentiation. LacZ expression in layer IV was accompanied by an increase in responsiveness of a subpopulation of layers II/III cells to spared whisker stimulation as determined by in vivo single-unit recording. Given that CREB is involved in the expression of plasticity in superficial layers (Glazewski et al., 1999), and yet CRE-mediated gene expression occurs in layer IV, it is likely that the molecular events initiating plasticity occur presynaptically to the cells that exhibit changes in their receptive field properties.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Neocórtex/fisiologia , Plasticidade Neuronal/fisiologia , Vias Aferentes/crescimento & desenvolvimento , Vias Aferentes/fisiologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Eletrofisiologia , Regulação da Expressão Gênica/fisiologia , Óperon Lac/genética , Camundongos , Camundongos Transgênicos , Neocórtex/crescimento & desenvolvimento , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Privação Sensorial/fisiologia , Regulação para Cima/fisiologia , Vibrissas/fisiologia , beta-Galactosidase/metabolismoAssuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Plasticidade Neuronal/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Ativação Enzimática/fisiologia , Memória/fisiologia , Vias Neurais , Sinapses/fisiologiaRESUMO
A program of stringently-regulated gene expression is thought to be a fundamental component of the circadian clock. Although recent work has implicated a role for E-box-dependent transcription in circadian rhythmicity, the contribution of other enhancer elements has yet to be assessed. Here, we report that cells of the suprachiasmatic nuclei (SCN) exhibit a prominent circadian oscillation in cAMP response element (CRE)-mediated gene expression. Maximal reporter gene expression occurred from late-subjective night to mid-subjective day. Cycling of CRE-dependent transcription was not observed in other brain regions, including the supraoptic nucleus and piriform cortex. Levels of the phospho-active form of the transcription factor CREB (P-CREB) varied as a function of circadian time. Peak P-CREB levels occurred during the mid- to late-subjective night. Furthermore, photic stimulation during the subjective night, but not during the subjective day, triggered a marked increase in CRE-mediated gene expression in the SCN. Reporter gene experiments showed that activation of the p44/42 mitogen-activated protein kinase signaling cascade is required for Ca2+-dependent stimulation of CRE-mediated transcription in the SCN. These findings reveal the CREB/CRE transcriptional pathway to be circadian-regulated within the SCN, and raise the possibility that this pathway provides signaling information essential for normal clock function.
Assuntos
Ritmo Circadiano , AMP Cíclico/genética , Proteínas Quinases Ativadas por Mitógeno , Núcleo Supraquiasmático/metabolismo , Animais , Cálcio/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Elementos Facilitadores Genéticos , Ativação Enzimática , Regulação da Expressão Gênica , Genes Reporter , Imuno-Histoquímica , Luz , Camundongos , Camundongos Transgênicos , Proteína Quinase 3 Ativada por Mitógeno , Fosfoproteínas/genética , Fosforilação , Transdução de SinaisRESUMO
Neuronal activity-dependent processes are believed to mediate the formation of synaptic connections during neocortical development, but the underlying intracellular mechanisms are not known. In the visual system, altering the pattern of visually driven neuronal activity by monocular deprivation induces cortical synaptic rearrangement during a postnatal developmental window, the critical period. Here, using transgenic mice carrying a CRE-lacZ reporter, we demonstrate that a calcium- and cAMP-regulated signaling pathway is activated following monocular deprivation. We find that monocular deprivation leads to an induction of CRE-mediated lacZ expression in the visual cortex preceding the onset of physiologic plasticity, and this induction is dramatically downregulated following the end of the critical period. These results suggest that CRE-dependent coordinate regulation of a network of genes may control physiologic plasticity during postnatal neocortical development.
Assuntos
Envelhecimento/fisiologia , Cálcio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , AMP Cíclico/metabolismo , Plasticidade Neuronal/fisiologia , Transcrição Gênica , Córtex Visual/fisiologia , Animais , Corpos Geniculados/fisiologia , Camundongos , Camundongos Transgênicos , Privação Sensorial/fisiologia , Transcrição Gênica/fisiologia , Visão Monocular/fisiologiaRESUMO
Specific forms of synaptic plasticity such as long-term potentiation (LTP) are modulated by or require increases in cAMP. The various adenylyl cyclase isoforms possess unique regulatory properties, and thus cAMP increases in a given cell type or tissue in response to converging signals are subject to the properties of the adenylyl cyclase isoforms expressed. In most tissues, adenylyl cyclase activity is stimulated by neurotransmitters or hormones via stimulatory G-protein (Gs)-coupled receptors and is inhibited via inhibitory G-protein (Gi)-linked receptors. However, in the hippocampus, stimulation of Gi-coupled receptors potentiates Gs-stimulated cAMP levels. This effect may be associated with the regulatory properties of adenylyl cyclase types 2 and 4 (AC2 and AC4), isoforms that are potentiated by the betagamma subunit of Gi in vitro. Although AC2 has been shown to be stimulated by betagamma in whole cells, reports describing the sensitivity of AC4 to betagamma in vivo have yet to emerge. Our results demonstrate that Gs-mediated stimulation of AC4 is potentiated by betagamma released from activated Gi-coupled receptors in intact human embryonic kidney (HEK) 293 cells. Furthermore, we show that the AC2 and AC4 proteins are expressed in the mouse hippocampal formation and that they colocalize with MAP2, a dendritic and/or postsynaptic marker. The presence of AC2 and AC4 in the hippocampus and the ability of each of these enzymes to detect coincident activation of Gs- and Gi-coupled receptors suggest that they may play a crucial role in certain forms of synaptic plasticity by coordinating such overlapping synaptic inputs.
Assuntos
Adenilil Ciclases/análise , Hipocampo/enzimologia , Isoenzimas/análise , Toxina Adenilato Ciclase , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Humanos , Imuno-Histoquímica , Rim/citologia , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Estimulação Química , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Although Ca2+-stimulated cAMP response element binding protein- (CREB-) dependent transcription has been implicated in growth, differentiation, and neuroplasticity, mechanisms for Ca2+-activated transcription have not been defined. Here, we report that extracellular signal-related protein kinase (ERK) signaling is obligatory for Ca2+-stimulated transcription in PC12 cells and hippocampal neurons. The sequential activation of ERK and Rsk2 by Ca2+ leads to the phosphorylation and transactivation of CREB. Interestingly, the Ca2+-induced nuclear translocation of ERK and Rsk2 to the nucleus requires protein kinase A (PKA) activation. This may explain why PKA activity is required for Ca2+-stimulated CREB-dependent transcription. Furthermore, the full expression of the late phase of long-term potentiation (L-LTP) and L-LTP-associated CRE-mediated transcription requires ERK activation, suggesting that the activation of CREB by ERK plays a critical role in the formation of long lasting neuronal plasticity.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Cálcio/fisiologia , Núcleo Celular/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Transcrição Gênica/fisiologia , Animais , Transporte Biológico/fisiologia , Sinalização do Cálcio , Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Hipocampo/citologia , Hipocampo/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Neurônios/fisiologia , Células PC12 , Fosforilação/efeitos dos fármacos , Ratos , Transdução de Sinais/fisiologiaRESUMO
Acute desensitization of olfactory signaling is a critical property of the olfactory system that allows animals to detect and respond to odorants. Correspondingly, an important feature of odorant-stimulated cAMP increases is their transient nature, a phenomenon that may be attributable to the unique regulatory properties of the olfactory adenylyl cyclase (AC3). AC3 is stimulated by receptor activation and inhibited by Ca2+ through Ca2+/calmodulin kinase II (CaMKII) phosphorylation at Ser-1076. Since odorant-stimulated cAMP increases are accompanied by elevated intracellular Ca2+, CaMKII inhibition of AC3 may contribute to termination of olfactory signaling. To test this hypothesis, we generated a polyclonal antibody specific for AC3 phosphorylated at Ser-1076. A brief exposure of mouse olfactory cilia or primary olfactory neurons to odorants stimulated phosphorylation of AC3 at Ser-1076. This phosphorylation was blocked by inhibitors of CaMKII, which also ablated cAMP decreases associated with odorant-stimulated cAMP transients. These data define a novel mechanism for termination of olfactory signaling that may be important in olfactory responses.
Assuntos
Adenilil Ciclases/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Neurônios/fisiologia , Odorantes , Mucosa Olfatória/fisiologia , Transdução de Sinais , Inibidores de Adenilil Ciclases , Adenilil Ciclases/química , Animais , Animais Recém-Nascidos , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Células Cultivadas , Cílios/efeitos dos fármacos , Cílios/fisiologia , AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Mucosa Olfatória/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosforilação , SerinaRESUMO
Marijuana consumption elicits diverse physiological and psychological effects in humans, including memory loss. Here we report that Delta9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, is toxic for hippocampal neurons. Treatment of cultured neurons or hippocampal slices with THC caused shrinkage of neuronal cell bodies and nuclei as well as genomic DNA strand breaks, hallmarks of neuronal apoptosis. Neuron death induced by THC was inhibited by nonsteroidal anti-inflammatory drugs, including indomethacin and aspirin, as well as vitamin E and other antioxidants. Furthermore, treatment of neurons with THC stimulated a significant increase in the release of arachidonic acid. We hypothesize that THC neurotoxicity is attributable to activation of the prostanoid synthesis pathway and generation of free radicals by cyclooxygenase. These data suggest that some of the memory deficits caused by cannabinoids may be caused by THC neurotoxicity.
Assuntos
Dronabinol/toxicidade , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Inibidores de Adenilil Ciclases , Animais , Ácido Araquidônico/metabolismo , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ativação Enzimática , Técnicas In Vitro , Neurônios/ultraestrutura , Fosfolipases A/efeitos dos fármacos , Prostaglandina-Endoperóxido Sintases/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Vitamina E/farmacologiaRESUMO
The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays an important regulatory role in developing and adult nervous systems. With the exception of the 5-HT3 receptor, all of the cloned serotonin receptors belong to the G protein-coupled receptor superfamily. Subtypes 5-HT6 and 5-HT7 couple to stimulation of adenylyl cyclases through Gs and display high affinities for antipsychotic and antidepressant drugs. In the brain, mRNA for 5-HT6 is found at high levels in the hippocampus, striatum, and nucleus accumbens. 5-HT7 mRNA is most abundant in the hippocampus, neocortex, and hypothalamus. To better understand how serotonin might control cAMP levels in the brain, we coexpressed 5-HT6 or 5-HT7A receptors with specific isoforms of adenylyl cyclase in HEK 293 cells. The 5-HT6 receptor functioned as a typical Gs-coupled receptor in that it stimulated AC5, a Gs-sensitive adenylyl cyclase, but not AC1 or AC8, calmodulin (CaM)-stimulated adenylyl cyclases that are not activated by Gs-coupled receptors in vivo. Surprisingly, serotonin activation of 5-HT7A stimulated AC1 and AC8 by increasing intracellular Ca2+. 5-HT also increased intracellular Ca2+ in primary neuron cultures. These data define a novel mechanism for the regulation of intracellular cAMP by serotonin.
Assuntos
Adenilil Ciclases/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Receptores de Serotonina/metabolismo , Linhagem Celular , Ativação Enzimática , Humanos , Fosfatidilinositóis/metabolismo , Serotonina/metabolismoRESUMO
Recent studies suggest that the CREB-CRE transcriptional pathway is pivotal in the formation of some types of long-term memory. However, it has not been demonstrated that stimuli that induce learning and memory activate CRE-mediated gene expression. To address this issue, we used a mouse strain transgenic for a CRE-lac Z reporter to examine the effects of hippocampus-dependent learning on CRE-mediated gene expression in the brain. Training for contextual conditioning or passive avoidance led to significant increases in CRE-dependent gene expression in areas CA1 and CA3 of the hippocampus. Auditory cue fear-conditioning, which is amygdala dependent, was associated with increased CRE-mediated gene expression in the amygdala, but not the hippocampus. These data demonstrate that learning in response to behavioral conditioning activates the CRE transcriptional pathway in specific areas of brain.
Assuntos
AMP Cíclico/fisiologia , Aprendizagem/fisiologia , Elementos de Resposta/fisiologia , Transcrição Gênica/fisiologia , Animais , Aprendizagem da Esquiva/fisiologia , Condicionamento Psicológico/fisiologia , AMP Cíclico/genética , Medo/fisiologia , Expressão Gênica/fisiologia , Hipocampo/fisiologia , Masculino , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos/genética , Elementos de Resposta/genéticaRESUMO
Although the circadian time-keeping properties of the suprachiasmatic nuclei (SCN) require gene expression, little is known about the signal transduction pathways that initiate transcription. Here we report that a brief exposure to light during the subjective night, but not during the subjective day, activates the p44/42 mitogen-activated protein kinase (MAPK) signaling cascade in the SCN. In addition, MAPK stimulation activates CREB (cAMP response element binding protein), indicating that potential downstream transcription factors are stimulated by the MAPK pathway in the SCN. We also observed striking circadian variations in MAPK activity within the SCN, suggesting that the MAPK cascade is involved in clock rhythmicity.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Ritmo Circadiano/fisiologia , Luz , Núcleo Supraquiasmático/fisiologia , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Ativação Enzimática/fisiologia , Ativação Enzimática/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologia , Núcleo Supraquiasmático/enzimologiaRESUMO
Gene expression regulated by the cAMP response element (CRE) has been implicated in synaptic plasticity and long-term memory. It has been proposed that CRE-mediated gene expression is stimulated by signals that induce long-term potentiation (LTP). To test this hypothesis, we made mice transgenic for a CRE-regulated reporter construct. We focused on long-lasting long-term potentiation (L-LTP), because it depends on cAMP-dependent protein kinase activity (PKA) and de novo gene expression. CRE-mediated gene expression was markedly increased after L-LTP, but not after decremental UP (D-LTP). Furthermore, inhibitors of PKA blocked L-LTP and associated increases in CRE-mediated gene expression. These data demonstrate that the signaling required for the generation of L-LTP but not D-LTP is sufficient to stimulate CRE-mediated transcription in the hippocampus.
Assuntos
Hipocampo/fisiologia , Potenciação de Longa Duração , Animais , Canais de Cálcio/fisiologia , AMP Cíclico/fisiologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Regiões Promotoras Genéticas , Proteínas Quinases/fisiologia , Receptores de AMP Cíclico/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de SinaisRESUMO
Type III adenylyl cyclase is stimulated by beta-adrenergic agonists and glucagon in vitro and in vivo, but not by Ca2+ and calmodulin. However, the enzyme is stimulated by Ca2+ and calmodulin in vitro when it is concomitantly activated by the guanyl nucleotide stimulatory protein Gs (Choi, E. J., Xia, Z., and Storm, D. R. (1992a) Biochemistry 31, 6492-6498). Here, we examined regulation of type III adenylyl cyclase by Gs-coupled receptors and intracellular Ca2+ in vivo. Surprisingly, intracellular Ca2+ inhibited hormone-stimulated type III adenylyl cyclase activity. Submicromolar concentrations of intracellular free Ca2+, which stimulated type I adenylyl cyclase, inhibited glucagon- or isoproterenol-stimulated type III adenylyl cyclase. Inhibition of type III adenylyl cyclase by intracellular Ca2+ was not mediated by Gi, cAMP-dependent protein kinase, or protein kinase C. However, an inhibitor of CaM kinases antagonized Ca2+ inhibition of the enzyme, and coexpression of constitutively activated CaM kinase II completely inhibited isoproterenol-stimulated type III adenylyl cyclase activity. We propose that Ca2+ inhibition of type III adenylyl cyclase may serve as a regulatory mechanism to attenuate hormone-stimulated cAMP levels in some tissues.