RESUMEN
Olfactory neuroepithelial cells in culture have been proposed as a model to study the physiopathology of psychiatric disorders and biomarker characterization for diagnosis. In patients with schizophrenia (SZ) and bipolar disorder (BD) diminished microtubule-associated proteins expression occurs, which might lead to aberrant microtubular organization and which in turn may affect Ca(2+) voltage-activated currents. The aim of this work was to characterize of microtubule organization as well as of the L-type Ca(2+) current in neuronal precursors obtained from nasal exfoliates of patients with SZ and BD. Microtubule organization was studied by immunofluorescence with a specific anti-III ß-tubulin antibody and by quantification of globular and assembled tubulin by Western blot. L-type current recording was performed by whole-cell patch-clamp technique and nifedipine superfusion. The results showed differential altered microtubular organization in neuronal precursors of SZ and BD. Short microtubules were observed in BD neurons, while extensive, unstained subcellular areas and disorganized microtubules were evident in SZ neuronal precursors. Patients with BD showed a decrease in amounts of tubulin in total homogenates and 40% decrease in the globular fraction. However, L-type current in BD was similar to that in healthy subjects (HS). In contrast, this current in SZ was 50% lower. These reduction in L-type current in SZ together with differential microtubule alterations are potential biomarkers that may differentiates SZ and BD.
Asunto(s)
Trastorno Bipolar , Canales de Calcio Tipo L , Calcio/metabolismo , Microtúbulos , Neuronas , Esquizofrenia , Biomarcadores/metabolismo , Trastorno Bipolar/metabolismo , Trastorno Bipolar/patología , Canales de Calcio Tipo L/metabolismo , Células Cultivadas , Humanos , Microtúbulos/metabolismo , Microtúbulos/patología , Neuronas/metabolismo , Neuronas/patología , Nifedipino , Técnicas de Placa-Clamp , Esquizofrenia/metabolismo , Esquizofrenia/patologíaRESUMEN
Brain imaging and histopathological studies suggest that neurodevelopmental anomalies play a key role in the etiology of schizophrenia (SZ) and bipolar disorder (BD). New neuron formation and maturation occur in human olfactory epithelium throughout life. Therefore, the olfactory epithelium has been proposed as a model to study alterations in neurodevelopment, particularly in some psychiatric diseases. However, former studies were done with olfactory epithelium biopsies taken post mortem or under anesthesia from patients with SZ and BD. In this work we have developed a new method to obtain viable neural precursors by exfoliation of the anterior region of the medial lateral turbinate of the nasal cavity from healthy controls, and ambulatory patients. Cells were propagated to establish neural precursor banks. Thawed cells showed cytoskeletal phenotypes typical of developing neurons. They also conserved the ability to differentiate in presence of 2mM dibutyril-cyclic adenosine monophosphate, and maintained voltage-operated Ca(2+) currents in culture. Moreover, proportions of neuronal maturation stages were maintained in cultured exfoliates obtained from SZ and BD patients. Data support that neural precursors obtained from a nasal exfoliate are an excellent experimental model to later approach studies on biomarkers, neural development and cellular alterations in the pathophysiology of SZ and BD.
Asunto(s)
Trastorno Bipolar/patología , Linaje de la Célula , Mucosa Nasal/citología , Neuronas , Esquizofrenia/patología , Células Madre , Adulto , Trastorno Bipolar/etiología , Linaje de la Célula/fisiología , Células Cultivadas , Femenino , Humanos , Masculino , Mucosa Nasal/patología , Neuronas/patología , Neuronas/fisiología , Esquizofrenia/etiología , Células Madre/patología , Células Madre/fisiología , Adulto JovenRESUMEN
AIMS: Nitric oxide (NO) is a free radical which reportedly causes damage to living cells. This study evaluated the damaging effect of NO and the protection of melatonin on the retina in vivo. METHODS: Female Wistar rats (230-250 g) received two intraperitoneal injections of either melatonin (5 mg/kg) or vehicle alone. After general anaesthesia, the animals received 1 microl intravitreal injections of 0.9% saline and 1 mM sodium nitroprusside (SNP) into the right eye and the left eye, respectively. The animals were divided into two groups and then sacrificed after 24 hours (day 1) and 96 hours (day 4). The mean inner retinal layer thickness (mIRLT), the number of retinas expressing hyperchromatic (HC) nuclei in the inner nuclear layer (INL) and the apoptotic ganglion cell detection were compared. RESULTS: After 1 day, SNP significantly increased the mIRLT by 45% (p = 0.004), initiated more INL nuclear HC expression (p = 0.01) and apoptotic nuclei (p<0.05) compared with the control eyes. Injection of melatonin ameliorated these changes. On day 4, SNP demonstrated similar effects in all parameters on the retina. After the injection of melatonin, both INL HC expression and apoptotic ganglion nuclei in the SNP treated eyes were similar to the controls but the mIRLT was significantly greater than in controls (p = 0.006). CONCLUSION: Uncontrolled NO elevation caused morphological and nuclear changes in the retina. Melatonin significantly suppressed the NO induced increase in mIRLT, INL HC expression, and apoptotic ganglion cells on day 1, but not after day 4. Melatonin may have a protective role in the NO elevated retina.
Asunto(s)
Antioxidantes/farmacología , Melatonina/farmacología , Óxido Nítrico/metabolismo , Retina/efectos de los fármacos , Animales , Apoptosis , Núcleo Celular/química , Cromatina , Proteínas del Ojo/análisis , Femenino , Inyecciones Intraperitoneales , Nitroprusiato/farmacología , Ratas , Ratas Wistar , Retina/química , Células Ganglionares de la Retina/fisiologíaRESUMEN
Progressive loss of neuronal cytoarchitecture is a major event that precedes neuronal death, both in neural aging and in neurodegenerative diseases. Cytoskeleton in neurodegenerative diseases is characterized by hyperphosphorylated tau assembled in neurofibrillary tangles. Tau protein promotes microtubule enlargement and its hyperphosphorylation inhibits tubulin assembly. Okadaic acid (OA) causes oxidative stress, tau hyperphosphorylation, and altered cytoskeletal organization similar to those observed in neurons of patients with dementia. Since melatonin acts by both enlarging microtubules and as a free-radical scavenger, in this work we studied the effects of melatonin on altered cytoskeletal organization induced by OA in N1E-115 neuroblastoma cells. Optic microscopy, morphometric analysis, and tubulin immunofluorescence staining of neuroblastoma cells incubated with 50 nM OA showed an intact microtubule network following the neurite profile similar to that observed in the vehicle-incubated cells when melatonin was added to the incubation media 2 h before OA. The melatonin effects on altered cytoskeletal organization induced by OA were dose-dependent and were not abolished by luzindole, the mt(1) melatonin antagonist receptor. Also, increased lipid peroxidation and augmented apoptosis in N1E-115 cells incubated with 50 nM OA were prevented by melatonin. The results support the hypothesis that melatonin can be useful in the treatment of neurodegenerative diseases.
Asunto(s)
Citoesqueleto/efectos de los fármacos , Melatonina/farmacología , Neuroblastoma/metabolismo , Ácido Ocadaico/farmacología , Estrés Oxidativo/fisiología , Animales , Citoesqueleto/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Neuroblastoma/tratamiento farmacológico , Toxina del Pertussis/farmacología , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
Melatonin, vitamin E and estrogen have been shown to exert neuroprotective effects against kainic acid (KA)-induced damage in the hippocampus. The aim of the present study was to examine the changes in potassium-evoked gamma-aminobutyric acid (GABA) release in the hippocampus of KA-treated rats and to test the possible protective effects of melatonin, vitamin E or estrogen. Following the treatment of mice with KA, a marked reduction in potassium-evoked [3H]GABA release was observed. Melatonin or estrogen prevented the reduction in potassium-evoked GABA release due to kainate administration. Vitamin E also exhibited some protective effect, but it was less than that provided by melatonin or estrogen. Melatonin, estrogen and, to a lesser extent, vitamin E reduce the physiological toxicity of KA. Since KA is believed to cause neuronal alterations via oxidative processes, it is assumed that the free radical scavenging and oxidative properties of melatonin, estrogen and vitamin E account for the protective effects of these agents.
Asunto(s)
Estradiol/análogos & derivados , Estradiol/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ácido Kaínico/antagonistas & inhibidores , Ácido Kaínico/toxicidad , Melatonina/farmacología , Vitamina E/farmacología , Ácido gamma-Aminobutírico/metabolismo , Animales , Antioxidantes/farmacología , Depuradores de Radicales Libres/farmacología , Hipocampo/lesiones , Masculino , Ratones , Ratones Endogámicos BALB C , Fármacos Neuroprotectores/farmacología , Potasio/farmacologíaRESUMEN
Previous reports have revealed that calmodulin antagonism by melatonin is followed by microtubule enlargements and neurite outgrowths in neuroblastoma N1E-115 cells. In addition, activation of protein kinase C (PKC) by this neurohormone is also followed by increased vimentin phosphorylation, and reorganization of vimentin intermediate filaments (IFs) in N1E-115 cells. In this work, we further characterize the activation of PKC by melatonin in neuroblastoma N1E-115 cells. We studied the Ca(2+)-dependent effects of melatonin on PKC activity and distribution of PKC-alpha in isolated N1E-115 cell IFs. Also, the effects of melatonin on PKC-alpha translocation in comparison to PKC-epsilon, were studied in intact N1E-115 cells. The results showed that both melatonin and the PKC agonist phorbol-12-myristate-13-acetate increased PKC activity in isolated IFs. The effects of the hormone were Ca(2+)-dependent, while those caused by the phorbol ester were produced with or without Ca(2+). Also, in isolated in situ IFs, the hormone changed the distribution of PKC-alpha. In intact N1E-115 cells, melatonin elicited PKC-alpha translocation and no changes were detected in PKC-epsilon. Phorbol-12-myristate-13-acetate modified the subcellular distribution of both PKC isoforms. The results showed that melatonin selectively activates the Ca(2+)-dependent alpha isoform of PKC and suggest that PKC-alpha activation by melatonin underlies IF rearrangements and participates in neurite formation in N1E-115 cells.
Asunto(s)
Isoenzimas/metabolismo , Melatonina/farmacología , Proteína Quinasa C/metabolismo , Animales , Línea Celular , Activación Enzimática , Filamentos Intermedios/enzimología , Filamentos Intermedios/metabolismo , Filamentos Intermedios/ultraestructura , Ratones , Microscopía Electrónica , Forbol 12,13-Dibutirato/metabolismo , Proteína Quinasa C-alfa , Proteína Quinasa C-epsilon , Transporte de Proteínas , Tritio , Células Tumorales CultivadasRESUMEN
It has been shown that melatonin through binding to calmodulin acts both in vitro and in vivo as a potent calmodulin antagonist. It is known that calmodulin antagonists both bind to the hydrophobic domain of Ca2+ activated calmodulin, and inhibit protein kinase C activity. In this work we explored the effects of melatonin on Ca2+ dependent protein kinase C activity in vitro using both a pure commercial rat brain protein kinase C, and a partially purified enzyme from MDCK and N1E-115 cell homogenates. The results showed that melatonin directly activated protein kinase C with a half stimulatory concentration of 1 nM. In addition the hormone augmented by 30% the phorbol ester stimulated protein kinase C activity and increased [3H] PDBu binding to the kinase. In contrast, calmodulin antagonists (500 microM) and protein kinase C inhibitors (100 microM) abolished the enzyme activity. Melatonin analogs tested were ineffective in increasing either protein kinase C activity or [3H] PDBu binding. Moreover, the hormone stimulated protein kinase C autophosphorylation directly and in the presence of phorbol ester and phosphatidylserine. The results show that besides the melatonin binding to calmodulin, the hormone also interacts with protein kinase C only in the presence of Ca2+. They also suggest that the melatonin mechanism of action may involve interactions with other intracellular hydrophobic and Ca2+ dependent proteins.
Asunto(s)
Encéfalo/enzimología , Melatonina/farmacología , Proteína Quinasa C/metabolismo , Animales , Calmodulina/antagonistas & inhibidores , Línea Celular , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Cinética , Forbol 12,13-Dibutirato/metabolismo , Unión Proteica , RatasRESUMEN
Recent evidence suggests that a melatonin (MEL) mechanism of action may be through modulation of Ca2+-activated calmodulin (CaM). MEL binds to CaM with a high affinity, and has been shown to act as a CaM antagonist. Among the CaM-dependent enzymes, Ca2+/Calmodulin-dependent protein kinase II (CaM-kinase II) is a particularly abundant enzyme in the nervous system. In the brain it phosphorylates a broad spectrum of substrates, thus modulating important neuronal functions. We describe the MEL effect on CaM-kinase II activity in vitro. CaM-kinase II was purified from rat brain by column chromatography, and identified by Western immunoblotting. CaM-kinase II activity was assessed in the presence of Ca2+/CaM by the kinase's ability to phosphorylate the synthetic substrate syntide-2 and by enzyme autophosphorylation. MEL inhibited CaM-kinase II activity, and enzyme autophosphorylation. Inhibition of the enzyme by 10(-9) M MEL was nearly of 30%. Trifluoperazine (10 microM), W7 (10 microM), and compound 48/80 (30 micrograms/ml), inhibited CaM-kinase II activity by 40%, 42%, and 93%, respectively. Both EGTA (5 mM) and MEL (10(-5) M) abolished autophosphorylation. The effect of MEL on CaM-kinase II activity was specific, since neither serotonin, N-acetylserotonin, nor 6-hydroxymelatonin inhibited its activity. Our results support the hypothesis that MEL acts as a CaM antagonist and cellular functions may be rhythmically regulated by MEL modulation of CaM-dependent protein phosphorylation.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Melatonina/farmacología , Animales , Encéfalo/enzimología , Calcio/metabolismo , Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Masculino , Fosforilación , RatasRESUMEN
Interaction of Entamoeba histolytica trophozoites with fibronectin (FN) induces reorganization of the actin cytoskeleton and an increase in proteolytic activities that results in the degradation of the bound protein. The binding is mediated by a 37-kDa FN "receptor" localized in the trophozoite surface and associated to the cytoskeleton. The intracellular signals triggered by the ligand-receptor interaction are not well understood but it is plausible that they drive the observed responses. To address this issue, the activation of protein kinase C (PKC) pathways by FN binding was explored. Stimulation with phorbol myristate acetate (PMA) or FN produced a rapid increase in the amebas adhesion to the substrate and local release of proteases. Two PKC inhibitors, H7 and staurosporine, reverted the PMA stimulus and inhibited the response induced by FN. Interaction with FN as well as treatment with PMA produced transient changes of F-actin levels susceptible to inhibition by H7. Furthermore, phosphorylation of amebic proteins was enhanced in response to FN binding and PMA, while the presence of the PKC inhibitor diminished their phosphorylation. Inositol triphosphate production was stimulated by the FN binding, and PKC activation and translation was registered in cell extracts obtained from the stimulated amebas. Our results suggest that PKC pathways are activated in amebas by information transduced as a result of trophozoite binding to FN.