RESUMO
Attention-deficit hyperactivity disorder (ADHD) is a common human neurobehavioral disorder that usually begins in early childhood. Methylphenidate (MPH) has been used extensively as a first-line medicine for the treatment of ADHD. Since ADHD is often diagnosed in early childhood and can persist for the entire lifespan, individuals may take MPH for many years. Given that in the course of one's lifetime a person may stop taking MPH for periods of time, or may implement lifestyle changes that may reduce the need for MPH entirely, it is important to understand how cessation of MPH affects the adult brain following long-term use of MPH. The blockage of the dopamine transporter (DAT) and the norepinephrine transporter (NET) by MPH may help with ADHD symptoms by boosting monoamine levels in the synapse. In the present study, microPET/CT was used to investigate possible neurochemical alterations in the cerebral dopamine system after cessation of long-term MPH administration in nonhuman primates. MicroPET/CT images were collected from adult male rhesus monkeys 6 months after they stopped receiving vehicle or MPH following 12 years of chronic treatment. The neurochemical status of brain dopaminergic systems was evaluated using the vesicular monoamine transporter 2 (VMAT2) ligand [18F]-AV-133 and a tracer for imaging dopamine subtype 2 (D2) and serotonin subfamily 2 (5HT2) receptors, [18F]-FESP. Each tracer was injected intravenously and ten minutes later microPET/CT images were obtained over 120 min. The binding potential (BP) of each tracer in the striatum was obtained using the Logan reference tissue model with the cerebellar cortex time activity curve (TAC) as an input function. Brain metabolism was also evaluated using microPET/CT images of [18F]-FDG. [18F]-FDG was injected intravenously, and ten minutes later, microPET/CT images were obtained over 120 min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum, and cerebellum were converted into standard uptake values (SUVs). Compared to the vehicle control group, the BPs of [18F] AV-133 and [18F]-FESP in the striatum were not significantly altered in MPH treated groups. Additionally, no significant differences were detected in the SUVs of [18F]-FDG in the MPH treated group compared with control. This study demonstrates that 6 months after cessation of long-term, chronic MPH treatment, there are no significant neurochemical or neural metabolic changes in the central nervous system (CNS) of non-human primates (NHPs) and suggests that microPET imaging is helpful in assessing the status of biomarkers of neurochemical processes linked to chronic CNS drug exposure. (Supported by NCTR).
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Estimulantes do Sistema Nervoso Central , Metilfenidato , Pré-Escolar , Adulto , Animais , Masculino , Humanos , Dopamina , Fluordesoxiglucose F18/uso terapêutico , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológicoRESUMO
Methylphenidate (MPH) is a psychostimulant approved by the FDA to treatment Attention-Deficit Hyperactivity Disorder (ADHD). MPH is believed to exert its pharmacological effects via preferential blockade of the dopamine transporter (DAT) and the norepinephrine transporter (NET), resulting in increased monoamine levels in the synapse. We used a quantitative non-invasive PET imaging technique to study the effects of long-term methylphenidate use on the central nervous system (CNS). We conducted microPET/CT scans on young adult male rhesus monkeys to monitor changes in the dopaminergic system. We used [18F] AV-133, a ligand for the vesicular monoamine transporter 2 (VMAT2), and [18F]FESP a ligand for the D2 and 5HT2 receptors. In this study we evaluated the effects if chronic MPH treatment in the nonhuman primates (NHP). Two-year-old, male rhesus monkeys were orally administered MPH diluted in the electrolyte replenisher, Prang, twice a day, five days per week (M-F) over an 8-year period. The dose of MPH was gradually escalated from 0.15 mg/kg initially to 2.5 mg/kg/dose for the low dose group, and 1.5 mg/kg to 12.5 mg/kg/dose for the high dose group (Rodriguez et al., 2010). Scans were performed on Mondays, about 60 h after their last treatment, to avoid the acute effects of MPH. Tracers were injected intravenously ten minutes before microPET/CT scanning. Sessions lasted about 120 min. The Logan reference tissue model was used to determine the Binding Potential (BP) of each tracer in the striatum with the cerebellar cortex time activity curve as an input function. Both MP treatment groups had a lower [18F] AV-133 BP, although this failed to reach statistical significance. MPH treatment did not have a significant effect on The BP of [18F] FESP in the striatum. Long-term administration of MPH did not significant change any of the marker of monoamine function used here. These data suggest that, despite lingering concerns, long-term use of methylphenidate does not negatively impact monoamine function. This study also demonstrates that microPET imaging can distinguish differences in binding potentials of a variety of radiotracers in the CNS of NHPs. This approach may provide minimally-invasive biomarkers of neurochemical processes associated with chronic exposure to CNS medications. (Supported by NCTR).
Assuntos
Encéfalo/efeitos dos fármacos , Proteínas da Membrana Plasmática de Transporte de Dopamina/efeitos dos fármacos , Metilfenidato/farmacologia , Fatores de Tempo , Animais , Estimulantes do Sistema Nervoso Central/administração & dosagem , Estimulantes do Sistema Nervoso Central/farmacologia , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Macaca mulatta , Metilfenidato/administração & dosagem , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/farmacologia , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada por Raios X/métodos , Proteínas Vesiculares de Transporte de Monoamina/efeitos dos fármacos , Proteínas Vesiculares de Transporte de Monoamina/metabolismoRESUMO
Interest in the recently discovered phenomenon of mitochondrial transfer between mammalian cells has gained momentum since it was first described in cell culture systems more than a decade ago. Mitochondria-targeting fluorescent dyes have been repurposed and are now widely used in these studies and in acute disease models, sometimes without due consideration of their limitations, while vectors containing mitochondrially-imported fluorescent proteins have complemented the use of mitochondria-targeting dyes. Genetic approaches that use mitochondrial DNA polymorphisms have also been used in some in vitro studies and in tumor models and are particularly useful where mtDNA is damaged or deleted. These approaches can also be used to study the long-term consequences of mitochondrial transfer such as in bone marrow and organ transplantation and in tumour biology where inherent mitochondrial damage is often a key feature. As research on intercellular mitochondrial transfer moves from cell culture into animal models and human diseases it will be important to understand the limitations of the various techniques in order to apply appropriate methodologies to address physiological and pathophysiological conditions.
Assuntos
Mitocôndrias/metabolismo , Polimorfismo Genético , Células A549 , Animais , Células Cultivadas , DNA Mitocondrial/genética , Corantes Fluorescentes/metabolismo , Marcadores Genéticos , Humanos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Modelos AnimaisRESUMO
Methylphenidate (MPH) is a psychostimulant commonly used for the treatment of Attention-Deficit Hyperactivity Disorder (ADHD). Since the long-term effects of this drug on the central nervous system (CNS) are not well understood, we conducted microPET/CT scans on young adult male rhesus monkeys (n=4/group) to gather information on brain metabolism using the uptake of [(18)F]Fluoro-2-deoxy-2-d-glucose (FDG) as a marker. Approximately two-year old, male rhesus monkeys were treated orally with MPH twice per day, five days per week (M-F) over a 6-year period. Subjects received MPH at either 2.5 or 12.5mg/kg/dose or vehicle (Prang). To minimize the acute effects of MPH on FDG uptake, microPET/CT scans were scheduled on Mondays before their first daily dosing of the week (approximately 68h since their last treatment). FDG (370±8.88MBq) was injected intravenously and 30min later microPET/CT images were obtained over 60min. Radiolabeled tracer accumulation in regions of interest (ROIs) in the prefrontal cortex, temporal cortex, striatum and cerebellum were converted into Standard Uptake Values (SUVs). Compared to the control group, the uptake of FDG in the cerebellum was significantly decreased in both the low and high dose groups. These preliminary data demonstrate that microPET imaging is capable of distinguishing differences in retention of FDG in the brains of NHPs treated chronically with MPH and suggests that this approach may provide a minimally invasive biomarker for exploring the effects of chronic MPH treatment on aspects of brain function.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Estimulantes do Sistema Nervoso Central/administração & dosagem , Metilfenidato/administração & dosagem , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada por Raios X , Animais , Fluordesoxiglucose F18/metabolismo , Macaca mulatta , MasculinoRESUMO
A number of naturally occurring isoforms of the tumour suppressor protein p53 have been discovered, which appear to have differing roles in tumour prevention or promotion. We are investigating the tumour-promoting activities of the Δ133p53 isoform using our mouse model of Δ133p53 (Δ122p53). Here, we report that tumours from Δ122p53 homozygous mice show evidence of invasion and metastasis and that Δ122p53 promotes migration though a 3-dimensional collagen matrix. We also show that Δ122p53 and Δ133p53 promote cell migration in scratch wound and Transwell assays, similar to the 'gain-of-function' phenotypes seen with mutant p53. Using the well-defined B16 mouse melanoma metastatic model, we show that Δ122p53 leads to faster generation of lung metastases. The increased migratory phenotypes are dependent on secreted factors, including the cytokine interleukin-6 and the chemokine CCL2. We propose that Δ122p53 (and Δ133p53) acts in a similar manner to 'gain-of-function' mutant p53 proteins to promote migration, invasion and metastasis, which may contribute to poor survival in patients with Δ133p53-expressing tumours.
Assuntos
Quimiocina CCL2/genética , Interleucina-6/genética , Neoplasias Pulmonares/genética , Melanoma Experimental/genética , Proteína Supressora de Tumor p53/genética , Animais , Movimento Celular/genética , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/patologia , Camundongos , Mutação , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Metástase Neoplásica , Isoformas de ProteínasRESUMO
Recent reports indicate that 6-12 h of ketamine anesthesia can trigger neuronal apoptosis in postnatal day (PND) 7 rats. In vitro, ex vivo, and confocal fluorescent imaging studies suggest that dansyl compounds can accumulate within the cytoplasm of the apoptotic cell. High-resolution positron emission tomography (microPET) imaging has been proposed as a minimally invasive method for detecting apoptosis in the rat brain. Compared with [(18)F]-labeled annexin V, which binds to externalized phosphatidylserine (PS) on the outer membrane of apoptotic cells, intracellular uptake of the dansylhydrazone of p-fluorobenzaldehyde (DFNSH) may lead to improved target-to-background contrast ratios. In this study, the effect of ketamine on the uptake and retention of [(18)F]-DFNSH in the rat brain was investigated using microPET imaging. On PND 7, rat pups in the experimental group were exposed, at 2-h intervals, to six subcutaneous injections of ketamine (20 mg/kg) and control rat pups received six injections of saline. On PND 35, [(18)F]-DFNSH (37 MBq) was injected into the tail vein of rats and microPET images were obtained over 2 h following the injection. Radiolabeled tracer accumulation in the region of interest (ROI) in the frontal cortex was converted into standard uptake values (SUVs). The radiotracer was quickly distributed into the brains of both ketamine- and saline-treated rats. Compared with the control group, the uptake of [(18)F]-DFNSH was significantly increased in the ROI, frontal cortex area of ketamine-treated rats. In addition, the wash-out duration of the tracer was prolonged in the ketamine-treated animals. This study demonstrates that microPET imaging is capable of distinguishing differences in retention of [(18)F]-DFNSH in ROI and suggests that this compound may serve as a minimally invasive biomarker of neuronal apoptosis in rodents.
Assuntos
Anestésicos Dissociativos/toxicidade , Benzaldeídos/farmacocinética , Encéfalo/diagnóstico por imagem , Ketamina/toxicidade , Neurônios/patologia , Tomografia por Emissão de Pósitrons/métodos , Animais , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Radioisótopos de Flúor/farmacocinética , Processamento de Imagem Assistida por Computador , Masculino , Neurônios/efeitos dos fármacos , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Sprague-Dawley , Distribuição TecidualRESUMO
Although the redox-active synthetic isoflavene, phenoxodiol, is in Phase 3 clinical trials for drug-resistant ovarian cancer, and in early stage clinical trials for prostate and cervical cancer, its primary molecular target is unknown. Nevertheless, phenoxodiol inhibits proliferation of many cancer cell lines and induces apoptosis by disrupting FLICE-inhibitory protein, FLIP, expression and by caspase-dependent and -independent degradation of the X-linked inhibitor of apoptosis, XIAP. In addition, phenoxodiol sensitizes drug-resistant tumour cells to anticancer drugs including paclitaxel, carboplatin and gemcitabine. Here, we investigate the effects of phenoxodiol on plasma membrane electron transport (PMET) and cell proliferation in human leukemic HL60 cells and mitochondrial gene knockout HL60rho(o) cells that exhibit elevated PMET. Phenoxodiol inhibited PMET by both HL60 (IC(50) 32 microM) and HL60rho(o) (IC(50) 70 microM) cells, and this was associated with inhibition of cell proliferation (IC(50) of 2.8 and 6.7 microM, respectively), pan-caspase activation and apoptosis. Unexpectedly, phenoxodiol also inhibited PMET by activated murine splenic T cells (IC(50) of 29 microM) as well as T cell proliferation (IC(50) of 2.5 microM). In contrast, proliferation of WI-38 cells and HUVECs was only weakly affected by phenoxodiol. These results indicate that PMET may be a primary target for phenoxodiol in tumour cells and in activated T cells.
Assuntos
Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Isoflavonas/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Doxorrubicina/farmacologia , Transporte de Elétrons/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Feminino , Citometria de Fluxo , Células HL-60 , Humanos , Concentração Inibidora 50 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Baço/citologia , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
In cardiac cells, Ca2+ signals appear as brief transients responsible for controlling both contraction and transcription. Information may be encoded in these digital signals through changes in both frequency and shape. An increase in Ca2+ signalling contributes to a process of phenotypic remodelling during hypertrophy. The increase in Ca2+ that drives the larger contractions may be responsible for switching on a second process of signalosome remodelling to down-regulate the Ca2+ signalling pathway. It is a change in the properties of the Ca2+ transient that seems to carry the information responsible for the remodelling of the cardiac gene transcription programme that leads first to hypertrophy and then to congestive heart failure.
Assuntos
Sinalização do Cálcio , Cardiomegalia/metabolismo , Animais , Cálcio/metabolismo , Cardiomegalia/genética , Insuficiência Cardíaca/metabolismo , Transcrição Gênica/genéticaRESUMO
Peloruside A (peloruside), a compound isolated from the marine sponge Mycale hentscheli , inhibits growth of human (HL-60) and mouse (32D-ras) myeloid leukemic cells, as well as non-transformed 32D cells. Using the MTT cell proliferation assay and trypan blue dye exclusion tests, little difference was seen in growth inhibition between 32D and 32D- ras cells; however, peloruside was more cytotoxic to the oncogene-transformed cells. Peloruside also blocked 32D- ras cells more readily in G2/M of the cell cycle, leading to apoptosis. Annexin-V/propidium iodide staining of 32D and 32D- ras cells showed that 1.6 microM peloruside induced significant cell death by 36 hours in 32D cells (16% survival), but to comparable levels as early as 14 hours in 32D- ras cells (11% survival). There was no evidence for activation of either of the initiator caspases-8 or -9 by 0.1 microM peloruside following 12 hours of exposure. Peloruside inhibited T cell proliferation and IL-2 and IFN gamma production in both the mixed lymphocyte reaction and following CD3 cross-linking, and this effect was shown to be a non-specific cytotoxic effect. It is concluded that peloruside preferentially targets oncogene-transformed cells over non-transformed cells by inducing transformed cells to undergo apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proliferação de Células/efeitos dos fármacos , Transformação Celular Neoplásica , Genes ras , Lactonas/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Anexina A5/metabolismo , Complexo CD3/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Reagentes de Ligações Cruzadas/farmacologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Células HL-60 , Humanos , Interferon gama/antagonistas & inibidores , Interleucina-2/antagonistas & inibidores , Teste de Cultura Mista de Linfócitos , Camundongos , Fatores de TempoRESUMO
Calcium regulates three different aspects of cardiac contraction. It drives pacemaker activity, excitation-contraction coupling and the transcriptional events that remodel the Ca(2+) signalling system in both health and disease.
Assuntos
Cálcio/metabolismo , Miocárdio/metabolismo , Transdução de Sinais , Potenciais de Ação , Animais , Relógios Biológicos , Humanos , Modelos Biológicos , Oscilometria , Transcrição GênicaRESUMO
Elementary Ca(2+) signals, such as "Ca(2+) puffs", which arise from the activation of inositol 1,4,5-trisphosphate receptors, are building blocks for local and global Ca(2+) signalling. We characterized Ca(2+) puffs in six cell types that expressed differing ratios of the three inositol 1,4,5-trisphosphate receptor isoforms. The amplitudes, spatial spreads and kinetics of the events were similar in each of the cell types. The resemblance of Ca(2+) puffs in these cell types suggests that they are a generic elementary Ca(2+) signal and, furthermore, that the different inositol 1,4,5-trisphosphate isoforms are functionally redundant at the level of subcellular Ca(2+) signalling. Hormonal stimulation of SH-SY5Y neuroblastoma cells and HeLa cells for several hours downregulated inositol 1,4,5-trisphosphate expression and concomitantly altered the properties of the Ca(2+) puffs. The amplitude and duration of Ca(2+) puffs were substantially reduced. In addition, the number of Ca(2+) puff sites active during the onset of a Ca(2+) wave declined. The consequence of the changes in Ca(2+) puff properties was that cells displayed a lower propensity to trigger regenerative Ca(2+) waves. Therefore, Ca(2+) puffs underlie inositol 1,4,5-trisphosphate signalling in diverse cell types and are focal points for regulation of cellular responses.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Linhagem Celular , Núcleo Celular/metabolismo , Regulação para Baixo , Histamina/farmacologia , Humanos , Imuno-Histoquímica , Receptores de Inositol 1,4,5-Trifosfato , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Gamma scintigraphy is often used to quantify deposition patterns from aerosol inhalers. The errors caused by scatter and tissue attenuation in planar Tc-99m gamma scintigraphy were investigated based on the data collected from four subjects in this study. Several error correction methods were tested. The results from two scatter correction methods, Jaszczak's method and factor analysis of dynamic sequences (FADS), were similar. Scatter accounted for 20% of raw data in the whole lung, 20% in the oropharynx, and 43% in the central airways and esophagus. Three attenuation correction methods were investigated and compared. These were: uniform attenuation correction (UAC), a known method used for inhalation drug imaging work; the broad-beam attenuation correction used for organ imaging in nuclear medicine; and a narrow-beam inhomogeneous tissue attenuation correction proposed in this study. The three methods differed significantly (p < 0.05), but all indicated that attenuation is a severe quantification problem. The narrow beam attenuation correction with scatter correction, showed that raw data underestimated tracer deposition by 44% in the lung, 137% in the oropharynx, and 153% in the trachea/esophageal region. To quantify aerosol lung deposition using planar scintigraphy even in relative terms, corrections are necessary. Much of the literature concerning quantified aerosol dose distributions measured by gamma scintigraphy needs to be interpreted carefully.
Assuntos
Aerossóis/farmacocinética , Anti-Inflamatórios/administração & dosagem , Anti-Inflamatórios/farmacocinética , Esôfago/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Orofaringe/efeitos dos fármacos , Cintilografia/normas , Tecnécio , Traqueia/efeitos dos fármacos , Triancinolona Acetonida/administração & dosagem , Triancinolona Acetonida/farmacocinética , Administração por Inalação , Adulto , Viés , Interpretação Estatística de Dados , Análise Fatorial , Humanos , Masculino , Pessoa de Meia-Idade , Nebulizadores e Vaporizadores , Tamanho da Partícula , Cintilografia/instrumentação , Cintilografia/métodos , Espalhamento de Radiação , Tecnécio/farmacocinética , Distribuição TecidualRESUMO
Ca2+ is a universal second messenger used to regulate a wide range of cellular processes such as fertilization, proliferation, contraction, secretion, learning and memory. Cells derive signal Ca2+ from both internal and external sources. The Ca2+ flowing through these channels constitute the elementary events of Ca2+ signalling. Ca2+ can act within milliseconds in highly localized regions or it can act much more slowly as a global wave that spreads the signal throughout the cell. Various pumps and exchangers are responsible for returning the elevated levels of Ca2+ back to the resting state. The mitochondrion also plays a critical role in that it helps the recovery process by taking Ca2+ up from the cytoplasm. Alterations in the ebb and flow of Ca2+ through the mitochondria can lead to cell death. A good example of the complexity of Ca2+ signalling is its role in regulating cell proliferation, such as the activation of lymphocytes. The Ca2+ signal needs to be present for over two hours and this prolonged period of signalling depends upon the entry of external Ca2+ through a process of capacitative Ca2+ entry. The Ca2+ signal stimulates gene transcription and thus initiates the cell cycle processes that culminate in cell division.
Assuntos
Sinalização do Cálcio/fisiologia , Animais , Divisão Celular , HumanosRESUMO
(S)-[18F]Fluorocarazolol is a radiopharmaceutical developed to quantitatively assess beta-adrenergic receptors in vivo via positron emission tomography imaging. Since radioactive metabolites of (S)-[18F]fluorocarazolol rapidly appear in the plasma, methods for conveniently and reliably evaluating plasma for (S)-[18F]fluorocarazolol content are required. Here we present methods and validation of an approach using commercial extraction cartridges that is faster and more convenient than an approach using internal-surface reverse-phase chromatography but yields comparable results.
Assuntos
Antagonistas Adrenérgicos beta/sangue , Carbazóis/sangue , Cromatografia Líquida de Alta Pressão/métodos , Propanolaminas/sangue , Compostos Radiofarmacêuticos/sangue , Sensibilidade e EspecificidadeRESUMO
Calcium (Ca(2+)) is a ubiquitous intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation. The ability of a simple ion such as Ca(2+) to play a pivotal role in cell biology results from the facility that cells have to shape Ca(2+) signals in space, time and amplitude. To generate and interpret the variety of observed Ca(2+) signals, different cell types employ components selected from a Ca(2+) signalling 'toolkit', which comprises an array of homeostatic and sensory mechanisms. By mixing and matching components from the toolkit, cells can obtain Ca(2+) signals that suit their physiology. Recent studies have demonstrated the importance of local Ca(2+) signals in defining the specificity of the interaction of Ca(2+) with its targets. Furthermore, local Ca(2+) signals are the triggers and building blocks for larger global signals that propagate throughout cells.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Potenciais de Ação , Animais , Canais de Cálcio Tipo T/metabolismo , Humanos , Transporte de Íons , Mitocôndrias/metabolismo , Músculos/citologia , Músculos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Neurotransmissores/metabolismo , Pâncreas/citologia , Pâncreas/metabolismoRESUMO
RATIONALE: s-Fluorocarazolol [(S)-FCZ] is the major positron emission tomography (PET) ligand currently used to visualize central beta-adrenergic receptors in vivo, although its pharmacology is incompletely known. OBJECTIVE: Our objective was to comprehensively characterize the in vitro pharmacology of (S)- and (R)-FCZ to determine its suitability for study of central and peripheral beta-adrenergic receptors. METHODS: We characterized the in vitro pharmacology of (S)-FCZ at 42 biogenic amine receptors and transporters in vitro using the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. RESULTS: As expected (R)- and (S)-FCZ had high affinities for beta-adrenergic receptors (Ki values=0.08-0.45 nM) and negligible affinities (Ki values>100 nM) for nearly all other tested receptors and transporters with the exception of the h5-HT1A receptor for which (S)-FCZ had high affinity (Ki=34 nM). Interestingly, (R)-FCZ had low affinity for the h5-HT1A receptor (Ki=342 nM). CONCLUSION: The high affinity of (S)-FCZ for the h5-HT1A receptor is not likely to interfere with studies of peripheral beta-adrenergic receptors, since 5-HT1A receptors are expressed at very low levels outside the central nervous system. Indeed, computer simulations predict that even at low ligand concentrations, 5-HT1A binding in brain regions like hippocampus are likely to be substantial. Thus, (S)-FCZ may not be a suitable PET ligand for studies of central nervous system beta-adrenergic receptors unless the contribution by 5-HT1A sites can be shown to be negligible.
Assuntos
Antagonistas Adrenérgicos beta/metabolismo , Carbazóis/metabolismo , Propanolaminas/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores de Serotonina/metabolismo , Tomografia Computadorizada de Emissão , Humanos , Ligantes , Receptores 5-HT1 de Serotonina , Proteínas Recombinantes/metabolismo , EstereoisomerismoRESUMO
PET centers without particle accelerators make clinical PET widely available at reduced cost. For myocardial perfusion tracers, these satellite PET centers are limited to generator- produced 82Rb(+) and 62Cu[PTSM]. Their limitations motivate a search for transportable alternatives. In search of new tracers we have synthesized several 18F-labeled amines and quaternary ammonium salts. Among them, 4-[18F]fluorotri-N-methylanilinium ([18F]FTMA) has flow-tracing properties. The compound is functional, but has properties that justify a continued search.
Assuntos
Hidrocarbonetos Fluorados/síntese química , Miocárdio/metabolismo , Animais , Inibidores da Colinesterase/farmacologia , Circulação Coronária , Radioisótopos de Flúor , Hidrocarbonetos Fluorados/farmacocinética , Camundongos , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
UNLABELLED: Planar gamma-scintigraphy is often used to quantify pulmonary deposition patterns from aerosol inhalers. The results are quite different from those obtained using 3-dimensional PET and SPECT. The purpose of this study was to characterize the effects of scatter and tissue attenuation on the distribution of radiolabeled aerosol as measured by planar scintigraphy using Monte Carlo simulations. This study also investigated the applicability of a few correction methods used in inhalation studies. METHODS: Body density maps were derived from CT scans. Regions of interest-lungs, major airways, and esophagus-were defined from the same CT volume. Two radioactivity source distribution patterns in the lung, uniform and nonuniform, were used. A Monte Carlo program, SIMIND, was used to generate anterior and posterior gamma-images of the composed inhalation distributions for 2 energy windows, photopeak (127-153 keV) and scatter (92-125 keV). The effects of scatter and attenuation were estimated on the basis of the imaging components separated from the simulation. A scatter correction method and 2 attenuation correction methods, all applied to inhalation scintigraphy, were evaluated using the simulated images. RESULTS: The amount of scatter ranges from 24% to approximately 29% in the lungs and from 29% to approximately 35% in the central (airway or esophagus) region on the planar images. Significant differences were found among regions and between source distributions (P < 0.05). The fraction k used for dual-energy-based scatter correction also varied and was found to be less than the commonly used k = 0.5. The simplified narrow-beam attenuation correction and the effective (broad-beam) correction methods were found to either under- or overcorrect the regional activities. CONCLUSION: The amount of scatter and tissue attenuation in the thorax region depends on source distribution and body attenuation. In applying planar scintigraphy for aerosol inhalation studies, it is difficult to obtain precise quantitative measurements because of the uncertainties associated with scatter and attenuation corrections. Accurate corrections require knowledge of both source and density distributions.
Assuntos
Aerossóis/farmacocinética , Câmaras gama , Pulmão/química , Pulmão/diagnóstico por imagem , Administração por Inalação , Adulto , Simulação por Computador , Humanos , Masculino , Pessoa de Meia-Idade , Método de Monte Carlo , Nebulizadores e Vaporizadores , Imagens de Fantasmas , Espalhamento de Radiação , Software , Tomografia Computadorizada de Emissão de Fóton Único/instrumentação , Tomografia Computadorizada por Raios XRESUMO
The marine sponge metabolites mycalamide A (mycalamide) and pateamine are extremely cytotoxic. While mycalamide has been shown to inhibit protein synthesis, the mechanism by which these compounds induce cell death is unknown. Using DNA laddering, Annexin-V staining, and morphological analysis, we demonstrate that both metabolites induce apoptosis in several different cell lines. Furthermore, both mycalamide and pateamine were more potent inducers of apoptosis in the 32D myeloid cell line after transformation with either the ras or bcr-abl oncogenes. This increased sensitivity was also observed in response to the protein synthesis inhibitors cycloheximide and puromycin, and cytosine-beta-D-arabinofuranoside (Ara-C), an inducer of DNA damage. We propose, therefore, that in 32D cells where Ras signalling has been altered either by constitutive expression of oncogenic ras or by Bcr/abl-mediated perturbation of upstream signalling events, increased susceptibility to apoptosis by a range of stimuli is conferred.
Assuntos
Apoptose , Compostos de Epóxi/farmacologia , Piranos/farmacologia , Tiazóis/farmacologia , Animais , Anexina A5/farmacologia , Sobrevivência Celular , Corantes/farmacologia , Cicloeximida/farmacologia , Citarabina/farmacologia , DNA/efeitos dos fármacos , Dano ao DNA , Eletroforese em Gel de Ágar , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Concentração Inibidora 50 , Macrolídeos , Modelos Químicos , Poríferos , Inibidores da Síntese de Proteínas/farmacologia , Puromicina/farmacologia , Sais de Tetrazólio/farmacologia , Azul Tripano/farmacologiaRESUMO
Using confocal imaging of Rhod-2-loaded HeLa cells, we examined the ability of mitochondria to sequester Ca(2+) signals arising from different sources. Mitochondrial Ca(2+) (Ca(2+)mit) uptake was stimulated by inositol 1,4,5-trisphosphate (InsP(3))-evoked Ca(2+) release, capacitative Ca(2+) entry, and Ca(2+) leaking from the endoplasmic reticulum. For each Ca(2+) source, the relationship between cytosolic Ca(2+) (Ca(2+)cyt) concentration and Ca(2+)mit was complex. With Ca(2+)cyt < 300 nm, a slow and persistent Ca(2+)mit uptake was observed. If Ca(2+)cyt increased above approximately 400 nm, Ca(2+)mit uptake accelerated sharply. For equivalent Ca(2+)cyt increases, the rate of Ca(2+)mit rise was greater with InsP(3)-evoked Ca(2+) signals than any other source. Spatial variation of the Ca(2+)mit response was observed within individual cells. Both the fraction of responsive mitochondria and the amplitude of the Ca(2+)mit response were graded in direct proportion to stimulus concentration. Trains of repetitive Ca(2+) oscillations did not maintain elevated Ca(2+)mit levels. Only low frequency Ca(2+) transients (<1/15 min) evoked repetitive Ca(2+)mit signals. Our data indicate that there is a lag between Ca(2+)cyt and Ca(2+)mit increases but that mitochondria will accumulate calcium when it is elevated over basal levels regardless of its source. Furthermore, in addition to the characteristics of Ca(2+) signals, Ca(2+) uniporter desensitization and proximity of mitochondria to InsP(3) receptors modulate mitochondrial Ca(2+) responses.