RESUMO
RATIONALE: Postsynaptic density-95 (PSD95) is a scaffolding protein that associates with voltage-gated, Shaker-type K(+) (KV1) channels and promotes the expression of KV1 channels in vascular smooth muscle cells of the cerebral (cVSMCs) circulation. However, the physiological role of PSD95 in mediating molecular signaling in cVSMCs is unknown. OBJECTIVE: We explored whether a specific interaction between PSD95 and KV1 channels enables protein kinase A phosphorylation of KV1 channels in cVSMCs to promote vasodilation. METHODS AND RESULTS: Rat cerebral arteries were used for analyses. A membrane-permeable peptide (KV1-C peptide) corresponding to the postsynaptic density-95, discs large, zonula occludens-1 binding motif in the C terminus of KV1.2α was designed as a dominant-negative peptide to disrupt the association of KV1 channels with PSD95. Application of KV1-C peptide to cannulated, pressurized cerebral arteries rapidly induced vasoconstriction and depolarized cVSMCs. These events corresponded to reduced coimmunoprecipitation of the PSD95 and KV1 proteins without altering surface expression. Middle cerebral arterioles imaged in situ through cranial window also constricted rapidly in response to local application of KV1-C peptide. Patch-clamp recordings confirmed that KV1-C peptide attenuates KV1 channel blocker (5-(4-phenylalkoxypsoralen))-sensitive current in cVSMCs. Western blots using a phospho-protein kinase A substrate antibody revealed that cerebral arteries exposed to KV1-C peptide showed markedly less phosphorylation of KV1.2α subunits. Finally, phosphatase inhibitors blunted both KV1-C peptide-mediated and protein kinase A inhibitor peptide-mediated vasoconstriction. CONCLUSIONS: These findings provide initial evidence that protein kinase A phosphorylation of KV1 channels is enabled by a dynamic association with PSD95 in cerebral arteries and suggest that a disruption of such association may compromise cerebral vasodilation and blood flow.
Assuntos
Artérias Cerebrais/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Potenciais da Membrana/fisiologia , Proteínas de Membrana/fisiologia , Superfamília Shaker de Canais de Potássio/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/efeitos dos fármacos , Proteína 4 Homóloga a Disks-Large , Inibidores Enzimáticos/farmacologia , Masculino , Modelos Animais , Técnicas de Patch-Clamp , Fosforilação/fisiologia , Ratos , Ratos Sprague-Dawley , Fluxo Sanguíneo Regional/efeitos dos fármacos , Fluxo Sanguíneo Regional/fisiologia , Vasoconstrição/fisiologia , Vasodilatação/fisiologiaRESUMO
Delayed engraftment and graft failure represent major obstacles to successful umbilical cord blood (UCB) transplantation. Herein, we evaluated the use of hyperbaric oxygen (HBO) therapy as an intervention to improve human UCB stem/progenitor cell engraftment in an immune deficient mouse model. Six- to eight-week old NSG mice were sublethally irradiated 24 hours prior to CD34⺠UCB cell transplant. Irradiated mice were separated into a non-HBO group (where mice remained under normoxic conditions) and the HBO group (where mice received 2 hours of HBO therapy; 100% oxygen at 2.5 atmospheres absolute). Four hours after completing HBO therapy, both groups intravenously received CD34⺠UCB cells that were transduced with a lentivirus carrying luciferase gene and expanded for in vivo imaging. Mice were imaged and then sacrificed at one of 10 times up to 4.5 months post-transplant. HBO treated mice demonstrated significantly improved bone marrow, peripheral blood, and spleen retention and subsequent engraftment. In addition, HBO significantly improved peripheral, spleen and bone marrow engraftment of human myeloid and B-cell subsets. In vivo imaging demonstrated that HBO mice had significantly higher ventral and dorsal bioluminescence values. These studies suggest that HBO treatment of NSG mice prior to UCB CD34⺠cell infusion significantly improves engraftment.
Assuntos
Subpopulações de Linfócitos B/citologia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Oxigenoterapia Hiperbárica , Animais , Antígenos CD34/genética , Antígenos CD34/imunologia , Subpopulações de Linfócitos B/imunologia , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Feminino , Expressão Gênica , Genes Reporter , Rejeição de Enxerto/imunologia , Humanos , Injeções Intravenosas , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos , Baço/citologia , Baço/imunologia , Transplante Heterólogo , Irradiação Corporal TotalRESUMO
Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is the most recently identified phosphoinositide, and its functions have yet to be fully elucidated. Recently, members of our muscle group have shown that PI(3,5)P2 plays an important role in skeletal muscle function by altering Ca(2+) homeostasis. Therefore, we hypothesized that PI(3,5)P2 may also modulate cardiac muscle contractility by altering intracellular Ca(2+) ([Ca(2+)](i)) in cardiac myocytes. We first confirmed that PI(3,5)P2 was present and increased by insulin treatment of cardiomyocytes via immunohistochemistry. To examine the acute effects of PI(3,5)P2 treatment, electrically paced left ventricular muscle strips were incubated with PI(3,5)P2. Treatment with PI(3,5)P2 increased the magnitude of isometric force, the rate of force development, and the area associated with the contractile waveforms. These enhanced contractile responses were also observed in MIP/Mtmr14(-/-) mouse hearts, which we found to have elevated levels of PI(3,5)P2. In cardiac myocytes loaded with fura-2, PI(3,5)P2 produced a robust elevation in [Ca(2+)](i). The PI(3,5)P2-induced elevation of [Ca(2+)](i) was not present in conditions free of extracellular Ca(2+) and was completely blocked by ryanodine. We investigated whether the phosphoinositide acted directly with the Ca(2+) release channels of the sarcoplasmic reticulum (ryanodine receptors; RyR2). PI(3,5)P2 increased [(3)H]ryanodine binding and increased the open probability (P(o)) of single RyR2 channels reconstituted in lipid bilayers. This strongly suggests that the phosphoinositide binds directly to the RyR2 channel. Thus, we provide inaugural evidence that PI(3,5)P2 is a powerful activator of sarcoplasmic reticulum Ca(2+) release and thereby modulates cardiac contractility.
Assuntos
Cálcio/metabolismo , Proteínas Musculares/metabolismo , Contração Miocárdica/fisiologia , Miócitos Cardíacos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Corantes Fluorescentes/farmacologia , Fura-2/farmacologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Bicamadas Lipídicas/metabolismo , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/citologia , Fosfatos de Fosfatidilinositol/genética , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Retículo Sarcoplasmático/genética , Retículo Sarcoplasmático/metabolismoRESUMO
Phosphoinositide (3,5)-bisphosphate [PI(3,5)P(2)] is a newly identified phosphoinositide that modulates intracellular Ca(2+) by activating ryanodine receptors (RyRs). Since the contractile state of arterial smooth muscle depends on the concentration of intracellular Ca(2+), we hypothesized that by mobilizing sarcoplasmic reticulum (SR) Ca(2+) stores PI(3,5)P(2) would increase intracellular Ca(2+) in arterial smooth muscle cells and cause vasocontraction. Using immunohistochemistry, we found that PI(3,5)P(2) was present in the mouse aorta and that exogenously applied PI(3,5)P(2) readily entered aortic smooth muscle cells. In isolated aortic smooth muscle cells, exogenous PI(3,5)P(2) elevated intracellular Ca(2+), and it also contracted aortic rings. Both the rise in intracellular Ca(2+) and the contraction caused by PI(3,5)P(2) were prevented by antagonizing RyRs, while the majority of the PI(3,5)P(2) response was intact after blockade of inositol (1,4,5)-trisphosphate receptors. Depletion of SR Ca(2+) stores with thapsigargin or caffeine and/or ryanodine blunted the Ca(2+) response and greatly attenuated the contraction elicited by PI(3,5)P(2). The removal of extracellular Ca(2+) or addition of verapamil to inhibit voltage-dependent Ca(2+) channels reduced but did not eliminate the Ca(2+) or contractile responses to PI(3,5)P(2). We also found that PI(3,5)P(2) depolarized aortic smooth muscle cells and that LaCl(3) inhibited those aspects of the PI(3,5)P(2) response attributable to extracellular Ca(2+). Thus, full and sustained aortic contractions to PI(3,5)P(2) required the release of SR Ca(2+), probably via the activation of RyR, and also extracellular Ca(2+) entry via voltage-dependent Ca(2+) channels.
Assuntos
Cálcio/metabolismo , Músculo Liso Vascular/metabolismo , Fosfatos de Fosfatidilinositol/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Aorta/metabolismo , Canais de Cálcio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
Dopamine-glutamate interactions in the neostriatum determine psychostimulant action, but the underlying molecular mechanisms remain elusive. Here we found that dopamine stimulation by cocaine enhances a heteroreceptor complex formation between dopamine D2 receptors (D2R) and NMDA receptor NR2B subunits in the neostriatum in vivo. The D2R-NR2B interaction is direct and occurs in the confined postsynaptic density microdomain of excitatory synapses. The enhanced D2R-NR2B interaction disrupts the association of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) with NR2B, reduces NR2B phosphorylation at a CaMKII-sensitive site (Ser1303), and inhibits NMDA receptor-mediated currents in medium-sized striatal neurons. Furthermore, the regulated D2R-NR2B interaction is critical for constructing behavioral responsiveness to cocaine. Our findings here uncover a direct and dynamic D2R-NR2B interaction in striatal neurons in vivo. This type of dopamine-glutamate integration at the receptor level may be responsible for synergistically inhibiting the D2R-mediated circuits in the basal ganglia and fulfilling the stimulative effect of psychostimulants.
Assuntos
Cocaína/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/fisiologia , Estimulantes do Sistema Nervoso Central/farmacologia , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Eletrofisiologia , Ácido Glutâmico/fisiologia , Imunoprecipitação , Locomoção/fisiologia , Masculino , Neostriado/citologia , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Fosforilação , Ligação Proteica , Ratos , Ratos WistarRESUMO
Trichloroacetaldehyde monohydrate [chloral hydrate (CH)] is a sedative/hypnotic that increases cerebral blood flow (CBF), and its active metabolite 2,2,2-trichloroethanol (TCE) is an agonist for the nonclassical two-pore domain K(+) (K(2P)) channels TREK-1 and TRAAK. We sought to determine whether TCE dilates cerebral arteries in vitro by activating nonclassical K(+) channels. TCE dilated pressurized and perfused rat middle cerebral arteries (MCAs) in a manner consistent with activation of nonclassical K(+) channels. Dilation to TCE was inhibited by elevated external K(+) but not by an inhibitory cocktail (IC) of classical K(+) channel blockers. Patch-clamp electrophysiology revealed that, in the presence of the IC, TCE increased whole-cell currents and hyperpolarized the membrane potential of isolated MCA smooth muscle cells. Heating increased TCE-sensitive currents, indicating that the activated channel was thermosensitive. Immunofluorescence in sections of the rat MCA demonstrated that, like TREK-1, TRAAK is expressed in the smooth muscle of cerebral arteries. Isoflurane did not, however, dilate the MCA, suggesting that TREK-1 was not functional. These data indicate that TCE activated a nonclassical K(+) channel with the characteristics of TRAAK in rat MCA smooth-muscle cells. Stimulation of K(+) channels such as TRAAK in cerebral arteries may therefore explain in part how CH/TCE increases CBF.
Assuntos
Etilenocloroidrina/análogos & derivados , Hipnóticos e Sedativos/farmacologia , Artéria Cerebral Média/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Canais de Potássio/fisiologia , Vasodilatadores/farmacologia , Animais , Etilenocloroidrina/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Isoflurano/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Artéria Cerebral Média/fisiologia , Músculo Liso Vascular/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/agonistas , Canais de Potássio/biossíntese , Ratos , Ratos Long-EvansRESUMO
Metabotropic glutamate receptors (mGluRs) are densely expressed in the limbic system of the mammalian brain. Increasing evidence suggests a critical role of mGluRs in the pathogenesis of various mental illnesses, including drug abuse and addiction. In this study, we investigated the effect of psychostimulant, cocaine, on protein expression of a specific mGluR subtype, mGluR8, in the rat forebrain in vivo. A rabbit antibody against the extracellular N-terminus of mGluR8 was developed to detect changes in mGluR8 proteins in immunoblot assays. With this antibody, we found that acute systemic injection of cocaine reduced mGluR8 protein levels in the striatum. The reduction of mGluR8 proteins was rapid and transient as it was induced 25min after cocaine injection and returned to the normal level by 6h. No significant change in mGluR8 protein levels in the prefrontal cortex and the hippocampus was observed following cocaine administration. These data demonstrate that protein expression of mGluR8 is subject to the modulation by dopamine stimulation. Acute exposure to cocaine results in a dynamic and region-specific downregulation of mGluR8 expression in the striatum.
Assuntos
Cocaína/administração & dosagem , Corpo Estriado/efeitos dos fármacos , Inibidores da Captação de Dopamina/administração & dosagem , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Metabotropic glutamate receptors (mGluRs) are G-protein-coupled receptors and are densely expressed in the forebrain of adult rats. Accumulative evidence suggests a critical role of mGluRs in the regulation of normal physiological activity of neurons and pathogenesis of mental illnesses such as schizophrenia, depression, and substance addiction. In this study, we investigated alterations in mGluR8 subtype mRNA expression in the rat forebrain in response to repeated intraperitoneal administration of amphetamine (twice daily for 12 days, 5mg/kg per injection) using quantitative in situ hybridization. We found that mGluR8 mRNA levels were profoundly increased in the dorsal (caudate putamen) and ventral (nucleus accumbens) striatum 1 day after the discontinuation of amphetamine treatments. Such increases were sustained up to 21 days of withdrawal. Increases in mGluR8 mRNAs were also found in the cerebral cortex, including the cingulate and sensory cortex but not the piriform cortex, at 1 and 21 days. These data demonstrate a positive response of mGluR8 in mRNA abundance in most forebrain regions to repeated stimulant exposure.
Assuntos
Transtornos Relacionados ao Uso de Anfetaminas/metabolismo , Anfetamina/farmacologia , Ácido Glutâmico/metabolismo , Prosencéfalo/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/genética , Transtornos Relacionados ao Uso de Anfetaminas/genética , Transtornos Relacionados ao Uso de Anfetaminas/fisiopatologia , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hibridização In Situ , Injeções Intraperitoneais , Masculino , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Prosencéfalo/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Recompensa , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Psychostimulants activate the Ras-mitogen-activated protein kinase (Ras-MAPK) cascade in the limbic reward circuit and thereby trigger a transcription-dependent mechanism underlying enduring synaptic plasticity related to addictive properties of drugs of abuse. The Ras-specific activator, Ras-guanine nucleotide-releasing factor (Ras-GRF), is predominantly expressed at synapses and is thought to actively regulate Ras-MAPK responses to changing synaptic signals. In this study, a possible influence of cocaine on Ras-GRF gene expression at the protein level in the rat striatum was investigated in vivo. A single systemic injection of cocaine induced an increase in Ras-GRF1 protein levels in both the dorsal (caudoputamen) and ventral (nucleus accumbens) striatum. The increase in Ras-GRF1 proteins was dose-dependent and was a delayed and transient event. In contrast to Ras-GRF1, a closely related Ras-GRF2 showed no change in its protein abundance following cocaine administration. These data identify the Ras activator, Ras-GRF1, although not Ras-GRF2, as a susceptible target to cocaine stimulation in striatal neurons.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , ras-GRF1/metabolismo , Animais , Relação Dose-Resposta a Droga , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Ratos , Ratos WistarRESUMO
The ionotropic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is densely distributed in the mammalian brain and is primarily involved in mediating fast excitatory synaptic transmission. Recent studies in both heterologous expression systems and cultured neurons have shown that the AMPA receptor can be phosphorylated on their subunits (GluR1, GluR2, and GluR4). All phosphorylation sites reside at serine, threonine, or tyrosine on the intracellular C-terminal domain. Several key protein kinases, such as protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and tyrosine kinases (Trks; receptor or nonreceptor family Trks) are involved in the site-specific regulation of the AMPA receptor phosphorylation. Other glutamate receptors (N-methyl-d-aspartate receptors and metabotropic glutamate receptors) also regulate AMPA receptors through a protein phosphorylation mechanism. Emerging evidence shows that as a rapid and short-term mechanism, the dynamic protein phosphorylation directly modulates the electrophysiological, morphological (externalization and internalization trafficking and clustering), and biochemical (synthesis and subunit composition) properties of the AMPA receptor, as well as protein-protein interactions between the AMPA receptor subunits and various intracellular interacting proteins. These modulations underlie the major molecular mechanisms that ultimately affect many forms of synaptic plasticity.
Assuntos
Plasticidade Neuronal , Receptores de AMPA/metabolismo , Transmissão Sináptica , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Fosforilação , Receptores de Glutamato/metabolismo , Receptores de Ácido Caínico/metabolismoRESUMO
Extracellular signals can regulate mitogen-activated protein kinase (MAPK) cascades through a receptor-mediated mechanism in postmitotic neurons of adult mammalian brain. Both ionotropic and metabotropic glutamate receptors (mGluRs) are found to possess such an ability in striatal neurons. NMDA and AMPA receptor signals seem to share a largely common route to MAPK phosphorylation which involves first activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) via Ca2+ influx, followed by subsequent induction of phosphoinositide 3-kinase (PI3-kinase). Through its lipid and protein kinase activity, active PI3-kinase may transduce signals to Ras-MAPK cascades via at least two distinct pathways. A novel, Ca(2+)-independent pathway is believed to mediate mGluR signals to Ras-MAPK activation. As an information superhighway between the surface membrane and the nucleus, Ras-MAPK cascades, through activating their specific nuclear transcription factor targets, are actively involved in the regulation of gene expression. Emerging evidence shows that MAPK-mediated genomic responses in striatal neurons to drug exposure contribute to the development of neuroplasticity related to addictive properties of drugs of abuse.
Assuntos
Ácido Glutâmico/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Neostriado/metabolismo , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Humanos , Plasticidade Neuronal/fisiologiaRESUMO
N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of subunits (NR1 and NR2A-D), and are enriched in the striatum. Receptor phosphorylation has recently been demonstrated on the NR1 subunit at three serine residues, 897, 896, and 890, which appear to correspond to the level of receptor activity. In this study, expression of phospho-specific NR1 subunits at serine 897 (pNR1S897), serine 896 (pNR1S896), or serine 890 (pNR1S890) in neurochemically identified neurons of the adult rat striatum was detected by using double-immunofluorescent labeling or combined in situ hybridization and immunohistochemistry. In both the dorsal and ventral striatum, pNR1S897 was expressed at high levels in projection neurons containing >55% dynorphin (striatonigral) and >90% enkephalin (striatopallidal) and in interneurons that were 100% positive for choline, >90% positive for parvalbumin, and >45% positive for somatostatin (co-containing neuropeptide Y and neuronal nitric oxide synthase). Low levels of pNR1S896 were present in a small portion of projection neurons (<15% for both populations of projection neurons) and were almost lacking in the three types of interneurons. Interestingly, pNR1S890 was exclusively expressed in most parvalbumin-containing interneurons (70-80%). Acute administration of a psychostimulant, amphetamine, increased the number of dynorphin-containing projection neurons and parvalbumin interneurons showing detectable levels of pNR1S896 and pNR1S890, respectively. These results demonstrate the distinct expression of phospho-NR1 subunits in different populations of striatal projection neurons and interneurons at variable levels in normal rats; they also demonstrate that phosphorylation of NR1, at least on serine 896 and 890 sites, is sensitive to drug exposure.
Assuntos
Anfetamina/farmacologia , Corpo Estriado/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Subunidades Proteicas/biossíntese , Receptores de N-Metil-D-Aspartato/biossíntese , Animais , Corpo Estriado/química , Corpo Estriado/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Interneurônios/química , Interneurônios/metabolismo , Masculino , Neurônios/química , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Metabotropic glutamate receptor 5 (mGluR5) is densely expressed in medium-sized spiny projection neurons of the rat striatum. Activation of mGluR5 increases intracellular Ca2+, resulting in Ca(2+)-dependent cellular responses. Acute administration of the psychostimulant amphetamine (AMPH) induces immediate early gene (IEG) expression in the striatum, which is considered an important molecular event for the development of striatal neuroplasticity related to the addictive properties of drugs of abuse. This study investigated the role of mGluR5 in the mediation of IEG expression in the rat striatum induced by a single dose of AMPH (4 mg/kg, i.p.) in vivo. We found that systemic administration of the mGluR5-selective antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) at a dose of 10 mg/kg, i.p. reduced AMPH-stimulated c-fos mRNA levels in the dorsal (caudoputamen) and ventral (nucleus accumbens) striatum as revealed by quantitative in situ hybridization. Similar results were observed in the three areas of cerebral cortex (cingulate, sensory, and piriform cortex). In contrast to c-fos mRNAs, AMPH-stimulated mRNA expression of another IEG, zif/268, was not significantly altered by the blockade of mGluR5 with MPEP in the entire striatum and the three areas of cortex. Treatment with MPEP alone had no effect on basal levels of c-fos and zif/268 mRNAs in the striatal and cortical areas. These results indicate that an mGluR5-dependent mechanism selectively contributes to c-fos expression in the striatum and cortex in response to acute exposure to AMPH.
Assuntos
Anfetamina/farmacologia , Corpo Estriado/efeitos dos fármacos , Genes Precoces/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/metabolismo , Regulação para Cima/efeitos dos fármacos , Transtornos Relacionados ao Uso de Anfetaminas/genética , Transtornos Relacionados ao Uso de Anfetaminas/metabolismo , Transtornos Relacionados ao Uso de Anfetaminas/fisiopatologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Genes Precoces/genética , Masculino , Proteínas Proto-Oncogênicas c-fos/genética , Piridinas/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptor de Glutamato Metabotrópico 5 , Regulação para Cima/genéticaRESUMO
Tumorigenicity studies often employ outbred nude mice, in the absence of direct evidence that this mixed genetic background will negatively affect experimental outcome. Here we show that outbred nude mice carry two different alleles of Pla2g2a, a genetic modifier of intestinal tumorigenesis in mice. Here, we identify previous unreported linked polymorphisms in the promoter, noncoding and coding sequences of Pla2g2a and show that outbred nude mice from different commercial providers are heterogeneous for this polymorphic Pla2g2a allele. This heterogeneity even extends to mice obtained from a single commercial provider, which display mixed Pla2g2a genotypes. Notably, we demonstrated that the polymorphic Pla2g2a allele affects orthotopic xenograft establishment of human colon cancer cells in outbred nude mice. This finding establishes a non-cell-autonomous role for Pla2g2a in suppressing intestinal tumorigenesis. Using in vitro reporter assays and pharmacological inhibitors, we show promoter polymorphisms and nonsense-mediated RNA decay (NMD) as underlying mechanisms that lead to low Pla2g2a mRNA levels in tumor-sensitive mice. Together, this study provides mechanistic insight regarding Pla2g2a polymorphisms and demonstrates a non-cell-autonomous role for Pla2g2a in suppressing tumors. Moreover, our direct demonstration that mixed genetic backgrounds of outbred nude mice can significantly affect baseline tumorigenicity cautions against future use of outbred mice for tumor xenograft studies.
Assuntos
Carcinogênese/genética , Fosfolipases A2 do Grupo II/genética , Polimorfismo Genético , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Alelos , Animais , Clonagem Molecular , Genótipo , Fosfolipases A2 do Grupo II/metabolismo , Células HCT116 , Humanos , Intestinos/patologia , Camundongos , Camundongos Nus , Degradação do RNAm Mediada por Códon sem Sentido , Plasmídeos/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
In treating cancer with clinically approved chemotherapies, the high systemic toxicity and lack of selectivity for malignant cells often result in an overall poor response rate. One pharmacological approach to improve patient response is to design targeted therapies that exploit the cancer milieu by reductively activating prodrugs, which results in the selective release of the free drug in the tumor tissue. Previously, we characterized prodrugs of seco-CBI-indole 2 (CBI-indole 2) designed to be activated in hypoxic tumor microenvironments, wherein the tumor maintains higher concentrations of "reducing" nucleophiles capable of preferentially releasing the free drug by nucleophilic attack on a weak N-O bond. Of these prodrugs, BocNHO-CBI-indole 2 (BocNHO) surpassed the efficacy of the free drug, CBI-indole 2, when examined in vivo in the murine L1210 leukemia model and demonstrated reduced toxicity suggesting a targeted or sustained release in vivo. Herein, we further examine the biological activity of the BocNHO prodrug in murine breast cancer, as well as human prostate and lung cancer cell lines, in vitro. Notably, BocNHO manifests potent antiproliferative and cytotoxic activity in all three tumor cell lines. However, in comparison to the activity observed in the murine cancer cell line, the human cancer cell lines were less sensitive, especially at early timepoints for cytotoxicity. Based on these findings, BocNHO was tested in a more clinically relevant orthotopic lung tumor model, revealing significant efficacy and reduced toxicity compared with the free drug. The data suggests that this pharmacological approach to designing targeted therapies is amenable to human solid tumors.
Assuntos
Antineoplásicos/farmacologia , Carbamatos/farmacologia , Indóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carbamatos/uso terapêutico , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Indóis/uso terapêutico , Camundongos , Camundongos Nus , Oxirredução , Pró-Fármacos/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Two novel cyclic N-acyl O-amino phenol prodrugs are reported as new members of a unique class of reductively cleaved prodrugs of the duocarmycin family of natural products. These prodrugs were explored with the expectation that they may be cleaved selectively within hypoxic tumor environments that have intrinsically higher concentrations of reducing nucleophiles and were designed to liberate the free drug without the release of an extraneous group. In vivo evaluation of the prodrug 6 showed that it exhibits extraordinary efficacy (T/C > 1500, L1210; 6/10 one year survivors), substantially exceeding that of the free drug, that its therapeutic window of activity is much larger, permitting a dosing ≥ 40-fold higher than the free drug, and yet that it displays a potency in vivo that approaches the free drug (within 3-fold). Clearly, the prodrug 6 benefits from either its controlled slow release of the free drug or its preferential intracellular reductive cleavage.
Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Indóis/administração & dosagem , Pró-Fármacos , Alquilação , Animais , Antibióticos Antineoplásicos/química , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , DNA/química , Preparações de Ação Retardada , Duocarmicinas , Indicadores e Reagentes , Indóis/química , Leucemia L1210/tratamento farmacológico , Camundongos , Pirrolidinonas/administração & dosagem , Pirrolidinonas/química , Espectrofotometria Ultravioleta , Estereoisomerismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
We sought to determine direct vascular effects of peroxisome proliferator-activated receptor alpha (PPARα) agonists using isolated mouse aortas and middle cerebral arteries (MCAs). The PPARα agonists GW7647, WY14643, and gemfibrozil acutely relaxed aortas held under isometric tension and dilated pressurized MCAs with the following order of potency: GW7647â«WY14643>gemfibrozil. Responses were endothelium-independent, and the use of PPARα deficient mice demonstrated that responses were also PPARα-independent. Pretreating arteries with high extracellular K(+) attenuated PPARα agonist-mediated relaxations in the aorta, but not in the MCA. In the aorta, the ATP sensitive potassium (K(ATP)) channel blocker glibenclamide also impaired relaxations whereas the other K(+) channel inhibitors, 4-aminopyridine and Iberiotoxin, had no effect. In aortas, GW7647 and WY14643 elevated cGMP levels by stimulating soluble guanylyl cyclase (sGC), and inhibition of sGC with ODQ blunted relaxations to PPARα agonists. In the MCA, dilations were inhibited by the protein kinase C (PKC) activator, phorbol 12,13-dibutyrate, and also by ODQ. Our results demonstrated acute, nonreceptor-mediated relaxant effects of PPARα agonists on smooth muscle of mouse arteries. Responses to PPARα agonists in the aorta involved K(ATP) channels and sGC, whereas in the MCA the PKC and sGC pathways also appeared to contribute to the response.
RESUMO
A unique heterocyclic carbamate prodrug of seco-CBI-indole(2) that releases no residual byproduct is reported as a new member of a class of hydrolyzable prodrugs of the duocarmycin and CC-1065 family of natural products. The prodrug was designed to be activated by hydrolysis of a carbamate releasing the free drug without the cleavage release of a traceable extraneous group. Unlike prior carbamate prodrugs examined that are rapidly cleaved in vivo, the cyclic carbamate was found to be exceptionally stable to hydrolysis under both chemical and biological conditions providing a slow, sustained release of the exceptionally potent free drug. An in vivo evaluation of the prodrug found that its efficacy exceeded that of the parent drug, that its therapeutic window of efficacy versus toxicity is much larger than the parent drug, and that its slow free drug release permitted the safe and efficacious use of doses 150-fold higher than the parent compound.
Assuntos
Antineoplásicos/metabolismo , Carbamatos/química , Desenho de Fármacos , Indóis/metabolismo , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Ciclopropanos/química , Duocarmicinas , Humanos , Hidrólise , Indóis/química , Indóis/farmacologia , Concentração Inibidora 50 , Camundongos , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Ras-guanine nucleotide-releasing factors (Ras-GRFs) are densely expressed in neurons of the mammalian brain. As a Ras-specific activator predominantly concentrated at synaptic sites, Ras-GRFs activate the Ras-mitogen-activated protein kinase (Ras-MAPK) cascade in response to changing synaptic inputs, thereby modifying a variety of cellular and synaptic activities. While the Ras-MAPK cascade in the limbic reward circuit is well-known to be sensitive to dopamine inputs, the sensitivity of its upstream activator (Ras-GRFs) to dopamine remains to be investigated. In this study, the response of Ras-GRFs in their protein expression to dopamine stimulation was evaluated in the rat striatum in vivo. A single systemic injection of the psychostimulant amphetamine produced an increase in Ras-GRF1 protein levels in both the dorsal (caudoputamen) and ventral (nucleus accumbens) striatum. The increase in Ras-GRF1 proteins was dose-dependent. The reliable increase was seen 2.5h after drug injection and returned to normal levels by 6h. In contrast to Ras-GRF1, protein levels of Ras-GRF2 in the striatum were not altered by amphetamine. In addition to the striatum, the medial prefrontal cortex is another forebrain site where amphetamine induced a parallel increase in Ras-GRF1 but not Ras-GRF2. No significant change in Ras-GRF1/2 proteins was observed in the hippocampus. These data demonstrate that Ras-GRF1 is a susceptible and selective target of amphetamine in striatal and cortical neurons. Its protein expression is subject to the modulation by acute exposure of amphetamine.
Assuntos
Anfetamina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , ras-GRF1/metabolismo , Anfetamina/administração & dosagem , Animais , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Injeções , Masculino , Atividade Motora/efeitos dos fármacos , Neostriado/citologia , Neostriado/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Putamen/efeitos dos fármacos , Putamen/metabolismo , Ratos , Ratos Wistar , Fatores de Tempo , Fatores ras de Troca de Nucleotídeo Guanina/metabolismoRESUMO
The glutamate receptor adaptor protein Homer is concentrated in the postsynaptic density of excitatory synapses and is critical for normal operation of synaptic transmission. In this study, we investigated the responsiveness of Homer family proteins to dopamine stimulation with the psychostimulant cocaine in rat striatal neurons both in vivo and in vitro. We found that a single dose of cocaine specifically induced a rapid and transient increase in protein levels of the Homer1a, but not Homer1b/c and Homer2a/b, isoforms in the striatum. This selective Homer1a induction was mediated primarily through activation of dopamine D1, but not D2, receptors. Both protein kinase A and Ca(2+)/calmodulin-dependent protein kinases are important for mediating the cocaine stimulation of Homer1a expression. At the transcriptional level, cAMP response element-binding protein serves as a prime transcription factor transmitting the signals derived from D1 receptors and associative pathways to the CaCRE sites within the Homer1a promoter. From a functional perspective, non-cross-linking Homer1a, once induced, competed with the cross-linking isoforms of Homer proteins (Homer1b/c and Homer2a/b) to uncouple the connection of group I metabotropic glutamate receptors (mGluRs) with inositol-1,4,5-triphosphate receptors. These results indicate that cocaine possesses the ability to stimulate Homer1a expression in striatal neurons through a specific synapse-to-nucleus pathway. Moreover, inducible Homer1a expression may represent a transcription-dependent mechanism underlying the dynamic regulation of submembranous macromolecular complex formation between group I mGluRs and their anchoring proteins.