RESUMEN
The progressing discovery of genetic variants associated with drug-related adverse events has raised expectations for pharmacogenetic tests to improve drug efficacy and safety. To further the use of pharmacogenetics in health care, tests with sufficient potential to improve efficacy and safety, as reflected by good clinical validity and population impact, need to be identified. The potential benefit of pharmacogenetic tests is often concluded from the strength of the association between the variant and the adverse event; measures of clinical validity are generally not reported. This paper describes measures of clinical validity and potential population health impact that can be calculated from association studies. We explain how these measures are influenced by the strength of the association and by the frequencies of the variant and the adverse event. The measures are illustrated using examples of testing for HLA-B*5701 associated with abacavir-induced hypersensitivity and SLCO1B1 c.521T>C (*5) associated with simvastatin-induced adverse events.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/genética , Didesoxinucleósidos/efectos adversos , Hipersensibilidad a las Drogas/genética , Pruebas Genéticas/métodos , Variación Genética/genética , Antígenos HLA-B/genética , Humanos , Farmacogenética/métodos , Simvastatina/efectos adversosRESUMEN
Drug-effect phenotypes in human lymphoblastoid cell lines recently allowed to identify CHL1 (cell adhesion molecule with homology to L1CAM), GAP43 (growth-associated protein 43) and ITGB3 (integrin beta 3) as new candidates for involvement in the antidepressant effect. CHL1 and ITGB3 code for adhesion molecules, while GAP43 codes for a neuron-specific cytosolic protein expressed in neuronal growth cones; all the three gene products are involved in synaptic plasticity. Sixteen polymorphisms in these genes were genotyped in two samples (n=369 and 90) with diagnosis of major depressive episode who were treated with antidepressants in a naturalistic setting. Phenotypes were response, remission and treatment-resistant depression. Logistic regression including appropriate covariates was performed. Genes associated with outcomes were investigated in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) genome-wide study (n=1861) as both individual genes and through a pathway analysis (Reactome and String databases). Gene-based analysis suggested CHL1 rs4003413, GAP43 rs283393 and rs9860828, ITGB3 rs3809865 as the top candidates due to their replication across the largest original sample and the STAR*D cohort. GAP43 molecular pathway was associated with both response and remission in the STAR*D, with ELAVL4 representing the gene with the highest percentage of single nucleotide polymorphisms (SNPs) associated with outcomes. Other promising genes emerging from the pathway analysis were ITGB1 and NRP1. The present study was the first to analyze cell adhesion genes and their molecular pathways in antidepressant response. Genes and biomarkers involved in neuronal adhesion should be considered by further studies aimed to identify predictors of antidepressant response.
Asunto(s)
Antidepresivos/uso terapéutico , Moléculas de Adhesión Celular Neuronal/genética , Adhesión Celular/genética , Trastorno Depresivo Mayor/tratamiento farmacológico , Trastorno Depresivo Mayor/genética , Biomarcadores/metabolismo , Moléculas de Adhesión Celular/genética , Trastorno Depresivo Mayor/metabolismo , Femenino , Proteína GAP-43/genética , Estudio de Asociación del Genoma Completo/métodos , Genotipo , Humanos , Integrina beta3/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Alzheimer's Disease (AD) is the most common cause of dementia in the elderly. Centenarians - reaching the age of >100 years while maintaining good cognitive skills - seemingly have unique biological features allowing healthy aging and protection from dementia. Here, we studied the expression of SIRT1 along with miR-132 and miR-212, two microRNAs known to regulate SIRT1, in lymphoblastoid cell lines (LCLs) from 45 healthy donors aged 21 to 105 years and 24 AD patients, and in postmortem olfactory bulb and hippocampus tissues from 14 AD patients and 20 age-matched non-demented individuals. We observed 4.0-fold (P = 0.001) lower expression of SIRT1, and correspondingly higher expression of miR-132 (1.7-fold; P = 0.014) and miR-212 (2.1-fold; P = 0.036), in LCLs from AD patients compared with age-matched healthy controls. Additionally, SIRT1 expression was 2.2-fold (P = 0.001) higher in centenarian LCLs compared with LCLs from individuals aged 56-82 years; while centenarian LCLs miR-132 and miR-212 indicated 7.6-fold and 4.1-fold lower expression, respectively. Correlations of SIRT1, miR-132 and miR-212 expression with cognitive scores were observed for AD patient-derived LCLs and postmortem AD olfactory bulb and hippocampus tissues, suggesting that higher SIRT1 expression, possibly mediated by lower miR-132 and miR-212, may protect aged individuals from dementia and is reflected in their peripheral tissues.
Asunto(s)
Enfermedad de Alzheimer/patología , Longevidad/genética , MicroARNs/metabolismo , Sirtuina 1/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Linfocitos B/citología , Linfocitos B/metabolismo , Estudios de Casos y Controles , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Bulbo Olfatorio/metabolismo , Adulto JovenRESUMEN
The current therapy success of depressive disorders remains in need of improvement due to low response rates and a delay in symptomatic improvement. Reliable functional biomarkers would be necessary to predict the individual treatment outcome. On the basis of the neurotrophic hypothesis of antidepressant's action, effects of antidepressant drugs on proliferation may serve as tentative individual markers for treatment efficacy. We studied individual differences in antidepressant drug effects on cell proliferation and gene expression in lymphoblastoid cell lines (LCLs) derived from patients treated for depression with documented clinical treatment outcome. Cell proliferation was characterized by EdU (5-ethynyl-2'-deoxyuridine) incorporation assays following a 3-week incubation with therapeutic concentrations of fluoxetine. Genome-wide expression profiling was conducted by microarrays, and candidate genes such as betacellulin-a gene involved in neuronal stem cell regeneration-were validated by quantitative real-time PCR. Ex vivo assessment of proliferation revealed large differences in fluoxetine-induced proliferation inhibition between donor LCLs, but no association with clinical response was observed. Genome-wide expression analyses followed by pathway and gene ontology analyses identified genes with different expression before vs after 21-day incubation with fluoxetine. Significant correlations between proliferation and gene expression of WNT2B, FZD7, TCF7L2, SULT4A1 and ABCB1 (all involved in neurogenesis or brain protection) were also found. Basal gene expression of SULT4A1 (P=0.029), and gene expression fold changes of WNT2B by ex vivo fluoxetine (P=0.025) correlated with clinical response and clinical remission, respectively. Thus, we identified potential gene expression biomarkers eventually being useful as baseline predictors or as longitudinal targets in antidepressant therapy.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Trastorno Depresivo/tratamiento farmacológico , Trastorno Depresivo/genética , Fluoxetina/uso terapéutico , Perfilación de la Expresión Génica , Expresión Génica/efectos de los fármacos , Estudio de Asociación del Genoma Completo , Adulto , Línea Celular , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Resultado del TratamientoRESUMEN
Major depression disorder (MDD) is the most widespread mental disorder. Selective serotonin reuptake inhibitors (SSRIs) are used as first-line MDD treatment but are effective in <70% of patients. Thus, biomarkers for the early identification of treatment-resistant (TR) MDD patients are needed for prioritizing them for alternative therapeutics. SSRI-induced inhibition of the growth of peripheral blood mononuclear cells (PBMCs) is mediated via their target, the serotonin transporter (SERT). Here, we examined whether antidepressant drug-induced inhibition of the growth of PBMCs differed between MDD patients and healthy controls. PBMCs from well-characterized 33 treatment-sensitive (TS) and 33 TR MDD patients, and 24 healthy volunteers were studied. Dose-dependent inhibition of PBMCs growth was observed for both the non-SSRI antidepressant mirtazapine and the SSRI antidepressant paroxetine. Significantly lower sensitivities to 20 µm paroxetine were observed in MDD compared with control PBMCs prior to treatment onset (13% and 46%, respectively; P<0.05). Following antidepressant drug treatment for 4 or 7 weeks, the ex vivo paroxetine sensitivity increased to control levels in PBMCs from TS but not from TR MDD patients. This suggests that the low ex vivo paroxetine sensitivity phenotype reflects a state marker of depression. A significantly lower expression of integrin beta-3 (ITGB3), a co-factor of the SERT, was observed in the PBMCs of MDD patients prior to treatment onset compared with healthy controls, and may explain their lower paroxetine sensitivity. Further studies with larger cohorts are required for clarifying the potential of reduced PBMCs paroxetine sensitivity and lower ITGB3 expression as MDD biomarkers.
Asunto(s)
Biomarcadores , Trastorno Depresivo Mayor/tratamiento farmacológico , Trastorno Depresivo Resistente al Tratamiento/tratamiento farmacológico , Mianserina/análogos & derivados , Monocitos/efectos de los fármacos , Paroxetina/uso terapéutico , Proteínas de Transporte de Serotonina en la Membrana Plasmática/efectos de los fármacos , Adulto , Proliferación Celular/efectos de los fármacos , Trastorno Depresivo Mayor/genética , Trastorno Depresivo Resistente al Tratamiento/genética , Femenino , Humanos , Integrina beta3/genética , Masculino , Mianserina/uso terapéutico , Persona de Mediana Edad , Mirtazapina , Fragmentos de PéptidosRESUMEN
Alzheimer's disease (AD) is the most frequent cause of dementia. Misfolded protein pathological hallmarks of AD are brain deposits of amyloid-ß (Aß) plaques and phosphorylated tau neurofibrillary tangles. However, doubts about the role of Aß in AD pathology have been raised as Aß is a common component of extracellular brain deposits found, also by in vivo imaging, in non-demented aged individuals. It has been suggested that some individuals are more prone to Aß neurotoxicity and hence more likely to develop AD when aging brains start accumulating Aß plaques. Here, we applied genome-wide transcriptomic profiling of lymphoblastoid cells lines (LCLs) from healthy individuals and AD patients for identifying genes that predict sensitivity to Aß. Real-time PCR validation identified 3.78-fold lower expression of RGS2 (regulator of G-protein signaling 2; P=0.0085) in LCLs from healthy individuals exhibiting high vs low Aß sensitivity. Furthermore, RGS2 showed 3.3-fold lower expression (P=0.0008) in AD LCLs compared with controls. Notably, RGS2 expression in AD LCLs correlated with the patients' cognitive function. Lower RGS2 expression levels were also discovered in published expression data sets from postmortem AD brain tissues as well as in mild cognitive impairment and AD blood samples compared with controls. In conclusion, Aß sensitivity phenotyping followed by transcriptomic profiling and published patient data mining identified reduced peripheral and brain expression levels of RGS2, a key regulator of G-protein-coupled receptor signaling and neuronal plasticity. RGS2 is suggested as a novel AD biomarker (alongside other genes) toward early AD detection and future disease modifying therapeutics.
Asunto(s)
Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Minería de Datos , Perfilación de la Expresión Génica , Expresión Génica/genética , Estudio de Asociación del Genoma Completo , Ovillos Neurofibrilares/genética , Placa Amiloide/genética , Proteínas RGS/genética , Anciano , Enfermedad de Alzheimer/diagnóstico , Enfermedad de Alzheimer/patología , Encéfalo/patología , Línea Celular , Biología Computacional , Diagnóstico Precoz , Estudios de Asociación Genética , Marcadores Genéticos/genética , Humanos , Masculino , Ovillos Neurofibrilares/patología , Fenotipo , Placa Amiloide/patologíaRESUMEN
The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [3H]-antagonist as well as of the labeled natural neurotransmitter, [3H]-acetylcholine [( 3H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [3H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [3H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [3H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [3H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms.
Asunto(s)
Acetilcolina/metabolismo , Bencilatos , Encéfalo/crecimiento & desarrollo , Oxotremorina/metabolismo , Parasimpatolíticos/metabolismo , Piperidinas/metabolismo , Receptores Muscarínicos/metabolismo , Envejecimiento , Animales , Encéfalo/metabolismo , Femenino , Masculino , Ratas , Ratas Endogámicas , Distribución Tisular , TritioRESUMEN
To assess the effect of hydrocarbon pneumonitis on the developing lung, we studied the pulmonary function of 17 asymptomatic children, 8 to 14 years after the initial insult. Fourteen of the 17 subjects (82%) had one or more pulmonary function abnormalities, the most frequent being a high volume of isoflow. Volume of isoflow, ratio residual volume to total lung capacity, slope of phase III, flow rates at 50% and 25% of vital capacity and 60% of the total lung capacity, one-second forced expiratory volume, and maximum midexpiratory flow rate differed significantly (P less than .05) from values in control groups. Closing volume and closing capacity were not significantly different. Residual abnormalities present in children after hydrocarbon pneumonitis can be explained on the basis of small airway obstruction and/or loss of elastic recoil. These children may be at risk for the development of chronic lung disease as adults when they are exposed to exogenous factors such as air pollution or smoking.
Asunto(s)
Hidrocarburos/efectos adversos , Pulmón/fisiopatología , Neumonía/complicaciones , Preescolar , Femenino , Humanos , Lactante , Masculino , Neumonía/inducido químicamente , Pruebas de Función RespiratoriaRESUMEN
Inherited antithrombin III (AT-III, heparin cofactor) deficiency is a rare condition, presenting with thrombotic disease in adult life. This paper reports an 8 months old South African Black male infant with multiple large vessel venous and arterial thromboses, and E. coli septicaemia. This was associated with an extremely low plasma AT-III level. Micronodular cirrhosis and intracytoplasmic hyaline globules in the liver cells were present. These globules were eosinophilic, and PAS-positive after diastase. They measured approximately 5 mu to 30 muin diameter, occurred singly in the liver cells and were located mainly in the periportal areas. The histological findings in the liver are similar to those observed in alpha 1-antitrypsin (AAT) deficiency in which the intracytoplasmic globules represent accumulation of altered AAT. Immunochemical studies carried out on formalin fixed tissue failed to detect cross reaction material with anti-alpha 1 antitrypsin or anti-AT III antiserum. This is the first case report of AT-III deficiency presenting in infancy. It is also the first case associated with distinctive liver pathology. The available data presented are insufficient to distinguish between an inborn defect and acquired caused of the severely depressed AT-III plasma level and the distinctive liver pathology.
Asunto(s)
alfa-Globulinas/deficiencia , Deficiencia de Antitrombina III , Tromboflebitis/complicaciones , Trombosis/complicaciones , Humanos , Hialina/análisis , Lactante , Hígado/análisis , Hígado/patología , Cirrosis Hepática/complicaciones , Masculino , Tromboflebitis/sangre , Tromboflebitis/patología , Trombosis/sangre , Trombosis/patologíaRESUMEN
Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AF102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the m1 AChR subtype. Notably, the selectivity of the new m1 agonists is reflected also by activation of select second messenger systems via distinct G-proteins. These compounds reflect a new pharmacological concept, tentatively defined as ligand-selective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimer's disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.
Asunto(s)
Acetilcolina/análogos & derivados , Acetilcolina/farmacología , Parasimpaticomiméticos/farmacología , Receptores Muscarínicos/fisiología , Transducción de Señal , Acetilcolina/síntesis química , Enfermedad de Alzheimer/fisiopatología , Animales , Células CHO , Cricetinae , Diseño de Fármacos , Humanos , Parasimpaticomiméticos/síntesis química , Receptores Muscarínicos/biosíntesis , Receptores Muscarínicos/efectos de los fármacos , TransfecciónRESUMEN
Epidemiologic studies suggest that the age-related decline in dehydroepiandrosterone (DHEA) levels may be associated with Alzheimer's disease (AD). Cholinergic markers also decline with age, and are associated with AD pathology. Activation of m1AChR-transfected PC12 cells (PC12M1) with cholinergic agonists results in secretion of Alzheimer's beta-amyloid precursor protein (APP) which in turn reduces beta-amyloid production. This study examined whether DHEA affects APP processing in m1AChR-transfected PC12 cells. DHEA treatment did not significantly alter basal or m1AChR-stimulated APP secretion. However, DHEA (0.1 microM) significantly diminished the desensitization of APP secretion in cells exposed to carbachol for 24 h. The effect of DHEA on APP processing is probably not related to up-regulation of m1AChR or increased m1AChR-activated phosphoinositide hydrolysis since these parameters did not change following DHEA treatment. These findings imply a possible involvement of DHEA in APP processing. Thus, the age-associated decline in DHEA levels may contribute to decreased APP secretion and a consecutive increase in beta-amyloid deposition, which in turn may play a role in the development of AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Deshidroepiandrosterona/farmacología , Receptores Muscarínicos/fisiología , Animales , Carbacol/farmacología , Agonistas Muscarínicos/farmacología , Células PC12 , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Tasa de Secreción/efectos de los fármacos , TransfecciónRESUMEN
The AF series compounds, AF102B and congeners of AF150(S), are functionally selective agonists for m1 muscarinic receptors (m1AChRs). This is shown in stable transfected CHO and PC12 cells (PC12M1) with m1m5AChRs and m1AChRs, respectively. AF102B and AF150(S) are partial agonists, but AF150, AF151, and AF151 (S) are full agonists in stimulating phosphoinositides hydrolysis or arachidonic acid release in these cells. Yet, all these compounds behave as antagonists when compared with carbachol in elevating cAMP levels. In PC12M1 cells, unlike carbachol, the AF series compounds induce only minimal to moderate neurite outgrowth. Yet, these agonists synergize strongly with NGF, which by itself mediates only a mild response. Stimulation of m1AChRs by AF102B, AF150(S) and AF151(S) in PC12M1 cells enhances secretion of beta/A4 amyloid precursor protein derivatives (APPs). The enhanced APPs secretion induced by AF102B is potentiated by NGF. AF102B also stimulates APPs secretion from rat cortical slices. Stimulation of m1AChR in PC12M1 cells with carbachol or AF102B decreases tau phosphorylation as indicated by specific tau-1 mAb and alkaline phosphatase treatment. Due to the above mentioned properties m1 agonists may be of unique value in delaying the progression of Alzheimer's disease (AD). The AF series compounds show a wide safety margin and improve memory and learning deficits in animal models for AD. There is a dearth of clinical reports on m1 agonists. These include studies on AF102B and xanomeline, another m1 selective agonist. We tested AF102B in escalating doses of 20, 40, 60 mg, tid, po, (each dose for 2 weeks) for a total of 10 weeks. This was a single-blind placebo-controlled, parallel-group study in patients with probable AD. AF102B was significantly effective at 40 and 60 mg, tid in the ADAS, ADAS-cognitive and ADAS-word recognition scales.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Agonistas Muscarínicos/uso terapéutico , Tiofenos , Animales , Ensayos Clínicos como Asunto , Proteínas de Unión al GTP/fisiología , Humanos , Ligandos , Agonistas Muscarínicos/química , Agonistas Muscarínicos/farmacología , Factores de Crecimiento Nervioso/farmacología , Células PC12/efectos de los fármacos , Células PC12/metabolismo , Fosforilación , Piridinas/química , Piridinas/uso terapéutico , Quinuclidinas/uso terapéutico , Ratas , Receptores Colinérgicos/fisiología , Transducción de Señal , Tiadiazoles/química , Tiadiazoles/uso terapéutico , Proteínas tau/metabolismoRESUMEN
Thrombin-like enzymatic activity was measured in mouse brain homogenates and slices by cleavage of a peptide substrate, N-p-Tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin. The activity was localized mainly to white matter. However, it was not affected by specific thrombin inhibitors, and was found to represent the sum of at least two enzyme activities, a prolyl endopeptidase and an aminopeptidase. By specifically inhibiting this endogenous activity in combination with exogenously added thrombin, mouse brain tissue was shown to express a capacity of thrombin inhibitory activity equivalent to 0.2 mU thrombin/mg brain tissue. The present study offers a simple and reliable method for measuring total thrombin inhibitory activity in brain.
Asunto(s)
Antitrombinas/farmacología , Química Encefálica/fisiología , Hemostáticos/metabolismo , Hirudinas/farmacología , Trombina/metabolismo , Aminopeptidasas/metabolismo , Animales , Encéfalo/enzimología , Fluorometría/métodos , Fluorometría/normas , Hemostáticos/antagonistas & inhibidores , Hemostáticos/farmacología , Masculino , Ratones , Ratones Endogámicos ICR , Prolil Oligopeptidasas , Reproducibilidad de los Resultados , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato , Trombina/antagonistas & inhibidores , Trombina/farmacologíaRESUMEN
The non-selective muscarinic agonist oxotremorine induces atropine-sensitive neurite outgrowth in PC12 cells stably transfected with m1 muscarinic receptors. In contrast, AF102B, an M1-selective muscarinic agonist, mediated minimal neurite outgrowth in these cells. In the presence of nerve growth factor (NGF) however, it induced atropine-sensitive neurite outgrowth in almost half the cell population. AF102B mediated phosphoinositide hydrolysis, but unlike carbachol, it did not stimulate cyclic AMP accumulation in these cells. These signals were not affected by NGF, indicating that they were not directly responsible for the cholinergic neurotrophic-like response. Our observations suggest that AF102B may improve neuronal responsiveness to neurotrophic factors, and thus may provide another beneficial aspect for treating Alzheimer's disease.
Asunto(s)
Factores de Crecimiento Nervioso/farmacología , Quinuclidinas/farmacología , Tiofenos , Animales , Atropina/farmacología , Carbacol/farmacología , AMP Cíclico/metabolismo , Células PC12 , Parasimpaticomiméticos/farmacología , RatasRESUMEN
The expression of muscarinic binding sites was examined in a collection of primary brain tumors of different cellular origins and various degrees of dedifferentiation, as compared to control specimens. Eleven gliogenous tumors were examined, all of which contained substantial amounts of muscarinic binding sites. Most of the other tumor types examined did not display detectable binding of [3H]N-methyl-4-piperidyl benzilate ([3H]4NMPB). Scatchard analysis indicated the existence of homogeneous antagonist sites in both normal forebrain and glioblastoma multiforme, with Kd values of 1.2 nM and 0.9 nM, respectively. The density of muscarinic binding sites varied between tumors from different patients, and also between specimens prelevated from different areas of the same tumor. This variability, as well as the average density of binding sites, appeared to be larger in highly malignant tumors than in less malignant ones. In contrast, the density of muscarinic receptors from control specimens was invariably high, but within the same order of magnitude. To test whether the muscarinic binding activity in the brain tumors is correlated to other cholinoceptive properties, cholinesterase activity was also examined. Individual data for density of [3H]4NMPB binding sites were then plotted against corresponding values of cholinesterase activity. The pattern of distribution of these values was clearly different in tumor specimens, when compared to that observed in samples derived from non-malignant brain. Our observations indicate that human brain cells of gliogenous origin are capable of expressing muscarinic binding sites, and that, if a correlation exists between muscarinic receptors and cholinesterase levels in gliogenous tumors, it differs from that of non-malignant brain tissue.
Asunto(s)
Astrocitoma/análisis , Neoplasias Encefálicas/análisis , Lóbulo Frontal , Glioblastoma/análisis , Neoplasias Meníngeas/análisis , Meningioma/análisis , Lóbulo Parietal , Receptores Muscarínicos/análisis , Lóbulo Temporal , Adulto , Anciano , Astrocitoma/enzimología , Neoplasias Encefálicas/enzimología , Neoplasias Cerebelosas/análisis , Colinesterasas/metabolismo , Femenino , Glioblastoma/enzimología , Humanos , Masculino , Neoplasias Meníngeas/enzimología , Persona de Mediana EdadRESUMEN
Activation of transfected muscarinic m1 acetylcholine receptors (m1AChR) has been linked to several signal transduction pathways which include phosphoinositide hydrolysis, arachidonic acid release and cAMP accumulation. In Chinese hamster ovary cells stably transfected with the rat m1AChR gene, carbachol elicited all three responses with EC50 values of 2.6, 3.8 and 76 microM, respectively. However, pilocarpine and the selective muscarinic agonist AF102B activated phosphoinositide hydrolysis (by 94 and 27% vs. carbachol, respectively), while antagonizing carbachol-mediated cAMP accumulation. Carbachol also activated (by 4-fold) adenylyl cyclase in membranes prepared from these cells, indicating independence of this signal from intracellular mediators. Moreover, carbachol and AF102B similarly elevated cytosolic Ca2+ in intact m1AChR-transfected cells. The ligand-selective cAMP accumulation, its independence from Ca2+ and the carbachol-activated adenylyl cyclase in membranes suggest that it represents an independent m1AChR-mediated signal, unrelated to phosphoinositide hydrolysis. Selective muscarinic ligands such as AF102B may independently activate distinct signalling pathways, which may be important for designing cholinergic replacement therapy for treating Alzheimer's disease.
Asunto(s)
Receptores Colinérgicos/fisiología , Transducción de Señal/fisiología , Tiofenos , Adenilil Ciclasas/análisis , Animales , Ácido Araquidónico/metabolismo , Células CHO , Calcio/análisis , Carbacol/farmacología , Células Cultivadas , Cricetinae , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/fisiología , Hidrólisis , Fosfatidilinositoles/metabolismo , Pilocarpina/farmacología , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Quinuclidinas/farmacología , Receptores Colinérgicos/genética , TransfecciónRESUMEN
The lesions of coronary atherosclerosis represent the result of a complex, multicellular, inflammatory-healing response in the coronary arterial wall. In vivo and in vitro cellular and molecular studies have suggested a role for tissue homocysteine in endothelial cell injury and adverse extra-cellular matrix remodeling. Gene polymorphisms in relation with numerous risk factors might increase the incidence of coronary artery disease (CAD). In this review we have focused on the correlations between plasma homocysteine levels, the incidence of cardiovascular disease and the cytosine-to-thymidine substitution at nucleotide 677 (C677T) of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, coding for a key enzyme in methionine-homocysteine metabolism. The role of the C677T MTHFR gene polymorphism in the causation of CAD is controversial. We reviewed 12 recent case-control studies comprising 5370 genotyped patients with CAD and 4961 genotyped participants without CAD. There was no significant difference between those with and without CAD in the frequency of the C677T polymorphism (34.9 vs 33.6%). The frequency of homozygous C677T polymorphism in these groups was 10.9 versus 12.8%, respectively, although there were some ethnic differences in the C677T MTHFR polymorphism. In the analysis of the 12 studies, the odds ratio of CAD associated with the TT genotype (homozygous C677T polymorphism) was 1.18. Only slightly higher plasma homocysteine levels were observed in participants with the val/val (TT) genotype (14.4+/-2.9 micro mol/L in TT genotype vs 11.1+/-1.9 and 11.9+/-2 micro mol/L in CC and CT genotype, respectively). In addition, the relation between homocysteine increase after methionine loading and MTHFR genotypes is also controversial. However, hyperhomocysteinemia because of the C677T MTHFR allele may be corrected with oral folic acid therapy. Further investigations on the relationships between MTHFR genotypes and the incidence of CAD should be based on larger samples, paying attention to the differences between various ethnic populations. Individual therapeutic strategies based on single nucleotide polymorphism may become increasingly important for preventive treatment against polygenic CAD.
Asunto(s)
Enfermedad de la Arteria Coronaria/genética , Homocisteína/sangre , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Polimorfismo Genético , Ensayos Clínicos como Asunto , Enfermedad de la Arteria Coronaria/sangre , Enfermedad de la Arteria Coronaria/epidemiología , Genotipo , Homocisteína/metabolismo , Humanos , Hiperhomocisteinemia/fisiopatología , Incidencia , Metilenotetrahidrofolato Reductasa (NADPH2)/metabolismo , Mutación Puntual , PrevalenciaRESUMEN
An in vitro model of dissociated cerebral cultures, prepared from prenatal 15-16-days rat fetuses, was used to further characterize the neurotoxic effects caused by the antibiotic ionophore lasalocid-X-537A. The damage caused by lasalocid (1-2 microM, 2-4 hr) included swelling of perikarya, followed by cytolysis of most neurons present in the cultures. The neuronal damage was dose-dependent, noticeable at concentrations above 0.5 microM, and was more pronounced in established cultures (14 days in vitro-DIV) than in younger ones (7 DIV). Unlike neurons, no damage was observed in glia and other non-neuronal cells present in the cultures by exposure to 2 microM lasalocid. Moreover, the drug was not toxic for cultures of rat astrocytes and C6 glioma cells. Another calcium ionophore A-23187 (calcimycin, 1 microM), destroyed both neuronal and non-neuronal cells within 1 hr. Ca2+ influx was increased by 140% in cultures exposed to lasalocid (1.5 microM). The lasalocid neurotoxic effects were neither inhibited by 10 microM nimodipine (a calcium channel antagonist) nor by 10 microM 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX)(a non-N-methyl-D-aspartic acid (NMDA) receptor antagonist), but were exclusively blocked by 10 microM MK-801 (a non-competitive NMDA receptor/channel antagonist). The neurotoxicity induced by lasalocid was further confirmed by measurements of lactate dehydrogenase (LDH) released into the media. Lasalocid (1.5 microM) induced the release of both LDH and arachidonic acid (AA) (by 8 and 4 fold of control values, respectively), and this was blocked by MK-801 but not by CNQX. These results are in according with the observations that activation of calcium influx through the NMDA receptor leads to activation of phospholipase A2 (PLA2) and release of AA. In contrast, MK-801 did not block the release of either LDH or AA mediated by the calcium ionophore A-23187 (1 microM) in these cultures. [3H]-MK-801 binding to washed rat cortical membranes, a measure of direct interaction with the NMDA receptor/channel complex, was not affected by lasalocid either alone or in the presence of glutamate and glycine. [3H]-D-aspartate release, a measure of excitatory amino acid (EAA) secretion mediated by NMDA receptor activation, was increased by lasalocid and could be blocked by MK-801. These observations suggest that lasalocid induces selective neurotoxicity, which involves the NMDA receptor/channel complex, possibly indirectly, resulted in elevated intracellular Ca2+ levels and the subsequent glutamate or aspartate release.
Asunto(s)
Corteza Cerebral/efectos de los fármacos , Lasalocido/toxicidad , Neurotoxinas/toxicidad , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ionóforos/toxicidad , RatasRESUMEN
Dehydroepiandrosterone (DHEA), the major secretory product of the human adrenal cortex, significantly declines with advanced age. We have previously demonstrated that DHEA prevents the reduction in non-amyloidogenic APP processing, following prolonged stimulation of the muscarinic receptor, in PC12 cells that express the ml acetylcholine-receptor. The present study examined whether this effect may be mediated via modulation of APP metabolism. It was found that DHEA treatment increases the content of membrane-associated APP holoprotein by 24%, and the accumulation of secreted APP in the medium by 63%. No increase in viable cell number nor in nonspecific protein production was observed in DHEA-treated cells. Thus, DHEA seems to increase specifically both APP synthesis and secretion. We propose that the age-associated decline in DHEA levels may be related to the pathological APP metabolism observed in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Deshidroepiandrosterona/farmacología , Precursor de Proteína beta-Amiloide/biosíntesis , Animales , Recuento de Células , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Indoles/farmacología , Maleimidas/farmacología , Células PC12 , Biosíntesis de Proteínas , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/metabolismo , Ratas , Transducción de SeñalRESUMEN
Dispersed cerebral cells prepared from 15-16-day mouse foetuses were cultured for 7 days and exposed to lasalocid for periods of 4, 18 or 48 hr. Cultures were examined by phase contrast microscopy and processed for scanning electron microscopy. Lasalocid (1 and 2 mum) induced neurotoxic effects that were evident already at 4 hr, including swelling of perikarya, followed by cytolysis of most neurons present in the cultures. During the following 18 hr virtually the entire neuronal population degenerated completely. Cultures exposed to a lower concentration of lasalocid (0.5 mum) for 48 hr showed only mild damage to some neurons, and at 0.2 mum no damage could be observed, even after several days of continuous exposure. Notably, glial and other non-neuronal cells were not damaged by exposure to 2 mum-lasalocid, and cell division resumed on returning the cultures to regular growth media. Similarly, lasalocid (2 mum) was not toxic to cultured rat astrocytes. These morphological observations were followed by measurements of (45)Ca(2+) influx in the dissociated cerebral cultures. Lasalocid (1 mum) induced (45)Ca(2+) influx of 40% above dimethyl sulphoxide control values. The voltage-sensitive calcium channel antagonists nimodipine (10 mum) and D-600 (50 mum) did not inhibit lasalocid-mediated (45)Ca(2+) influx, whereas MK-801 [10 mum; a non-competitive N-methyl-d-aspartate (NMDA) receptor/channel antagonist] exclusively blocked this influx and prevented cytotoxic damage. Conversely, NMDA and glutamate, which by themselves mediated (45)Ca(2+) influx in these cultures, both potentiated lasalocid-induced (45)Ca(2+) influx and cellular damage. These observations clearly demonstrate the selective neurotoxicity of lasalocid to cultured cerebral neurons, and may imply involvement of the NMDA receptor/channel in lasalocid-mediated neurotoxicity.