RESUMEN
Schizophrenia (SZ) and Bipolar Disorder (BD) are highly inheritable chronic mental disorders with a worldwide prevalence of around 1%. Despite that many efforts had been made to characterize biomarkers in order to allow for biological testing for their diagnoses, these disorders are currently detected and classified only by clinical appraisal based on the Diagnostic and Statistical Manual of Mental Disorders. Olfactory neuroepithelium-derived neuronal precursors have been recently proposed as a model for biomarker characterization. Because of their peripheral localization, they are amenable to collection and suitable for being cultured and propagated in vitro. Olfactory neuroepithelial cells can be obtained by a non-invasive brush-exfoliation technique from neuropsychiatric patients and healthy subjects. Neuronal precursors isolated from these samples undergo in vitro the cytoskeletal reorganization inherent to the neurodevelopment process which has been described as one important feature in the etiology of both diseases. In this paper, we will review the current knowledge on microtubular organization in olfactory neurons of patients with SZ and with BD that may constitute specific cytoskeletal endophenotypes and their relation with alterations in L-type voltage-activated Ca(2+) currents. Finally, the potential usefulness of neuronal precursors for pharmacological screening will be discussed.
Asunto(s)
Trastorno Bipolar/metabolismo , Microtúbulos/patología , Neuronas Receptoras Olfatorias/citología , Esquizofrenia/metabolismo , Biomarcadores/metabolismo , Trastorno Bipolar/patología , Canales de Calcio Tipo L/metabolismo , Humanos , Microtúbulos/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Neuronas Receptoras Olfatorias/patología , Esquizofrenia/patologíaRESUMEN
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
The cytoskeleton plays a key role in maintaining the highly asymmetrical shape and structural polarity of neurons that are essential for neuronal physiology. Cytoskeletal reorganization plays a key role in neuritogenesis. In neurodegenerative diseases, the cytoskeleton is abnormally assembled and impairment of neurotransmission occurs. In Alzheimer's disease, abundant amyloid plaques and neurofibrillary tangles constitute the two major neuropathologic alterations present in the brain. Neurofibrillary tangles are formed of paired helical filaments consisting nearly entirely of the microtubule-associated protein tau. Under normal conditions tau binds to microtubules, stabilizing neuron structure and integrity. Hyperphosphorylation of tau is assumed to be the cause of formation of paired helical filaments. Another example of cytoskeletal abnormalities present in neurodegenerative diseases are the Lewy bodies considered as cytopathologic markers of Parkinson's disease. Lewy bodies are constituted of tubulin, MAP1, and MAP2. Neuronal shape, loss of dendrites and spines, as well as irregular distribution of neuronal elongations occur in specific brain areas of schizophrenic patients. Increase in non-phosphorylated MAP2 and MAP1B at hippocampus has been suggested as responsible for somatodendritic and cytoarchitectural abnormalities found in schizophrenia. In addition, neurofibrillary tangles are more frequent among schizophrenic patients who received pharmacologic antipsychotic treatment. Cumulative evidence suggests that neurodegenerative diseases and psychiatric illnesses are associated with cytoskeletal alterations in neurons that, in turn, loose synaptic connectivity and the ability to transmit incoming axonal information to the somatodendritic domain. We will review evidence supporting that the neuronal cytoskeleton is disrupted in neurodegenerative and some psychiatric diseases, and therefore could be a target for drug therapy. In addition, current data indicating that melatonin, a hormone secreted by the pineal gland, promotes neuritogenesis through cytoskeletal rearrangements and in addition to the potential therapeutic use of melatonin in neurodegenerative diseases will be discussed.
Asunto(s)
Encéfalo/metabolismo , Citoesqueleto/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Neuronas/metabolismo , Esquizofrenia/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Citoesqueleto/efectos de los fármacos , Humanos , Cuerpos de Lewy/metabolismo , Cuerpos de Lewy/patología , Melatonina/farmacología , Melatonina/uso terapéutico , Proteínas Asociadas a Microtúbulos/metabolismo , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/fisiopatología , Ovillos Neurofibrilares/metabolismo , Ovillos Neurofibrilares/patología , Neuronas/efectos de los fármacos , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/fisiopatologíaRESUMEN
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
The interaction between melatonin and calmodulin was explored. Calmodulin cell levels in MDCK and N1E-115 cells cultured with 10(-9) M melatonin were increased after 3 days but decreased after 6 days. Melatonin inhibited calmodulin-dependent phosphodiesterase and when either melatonin or [3H]melatonin was preincubated with calmodulin and separated by electrophoresis, comigration of calmodulin with the radioactivity as well as modification of the Ca2+ calmodulin shift were observed. The results point out that one of the mechanisms of action of melatonin is a calmodulin-melatonin interaction.
Asunto(s)
Calmodulina/metabolismo , Melatonina/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Animales , Calmodulina/biosíntesis , Línea Celular , Perros , ElectroforesisRESUMEN
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
Studies in melatonin mechanism of action have suggested that one of them could be the binding of the hormone to calmodulin. We assessed calmodulin-melatonin binding by combining liposome incorporation of calmodulin with separation of free and bound 3H-Melatonin by a rapid ultrafiltration method. Specific binding to calmodulin was saturable, reversible, Ca(++)-dependent, ligand selective, and showed high affinity. Saturation as well as association-dissociation studies revealed that 3H-Melatonin binds to a single site on the calmodulin molecule with a Kd of 188 pM and a total binding capacity Bmax of 35 pM/ug of calmodulin. Displacement experiments showed that the relative order of potency of some compounds for inhibition of 3H-Melatonin was as follows: Melatonin > 6-chloromelatonin > 6-hydroxymelatonin > luzindole > trifluoperazine. The results explain our previously reported melatonin effects such as cytoskeletal rearrangements, inhibition of calmodulin dependent phosphodiesterase activity as well as the modification of Ca(++)-calmodulin electrophoretic mobility. The high affinity of melatonin binding to calmodulin suggests that the hormone is able to modulate cell activity by intracellularly binding to calmodulin at physiologically ranges. Melatonin-calmodulin binding could modulate many intracellular Ca++ functions and thus, the set-point for cell activity will follow the rhythmic circulating levels of the pineal hormone. Moreover, since calmodulin and melatonin are phylogenetically well preserved compounds, their interaction may represent a primary mechanism for both the regulation and the synchronization of cell physiology.
Asunto(s)
Calmodulina/metabolismo , Melatonina/metabolismo , Animales , Calcio/metabolismo , Calmodulina/antagonistas & inhibidores , Melatonina/antagonistas & inhibidores , Unión Proteica , Ovinos , TritioRESUMEN
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
Cell migration and adhesion are cytoskeleton- dependent functions that play a key role in epithelial physiology. Specialized epithelial cells in water transport have specific microfilament rearrangements that make these cells adopt a polyhedral shape, forming a sealed monolayer which functions as permeability barrier. Also, specific polarized microfilament phenotypes are formed at the front and the rear of migratory epithelial cells. In pathological processes such as cancer, increased migration occurs in invasive cells driven by the formation of polarized and differential microfilament phenotypes. Melatonin, the main product secreted by the pineal gland during dark phase of the photoperiod, acts as a cytoskeletal modulator in normal and cancer cells. In this paper we will summarize evidence supporting that melatonin acts as a microfilament modulator in epithelial MDCK cells, and we will describe its effects on cytoskeleton organization involved in the mechanism by which melatonin synchronizes water transport. In addition, we will review recent data that indicate that melatonin is able to switch microfilament phenotypes in MCF-7 human mammary cancer cells, from invasive migratory cells to dormant microfilament phenotypes that occur in non- migratory cells. Moreover, we will discuss the implications of the cytoskeleton as therapeutic target for cancer cells.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Adhesión Celular/fisiología , Movimiento Celular/fisiología , Células Epiteliales/metabolismo , Melatonina/metabolismo , Invasividad Neoplásica , Animales , Citoesqueleto/metabolismo , Humanos , Neoplasias/metabolismo , FenotipoRESUMEN
Melatonin enters cells and causes cytoskeletal rearrangements in unicellular organisms, plants and vertebrates. This pineal secretory product causes microtubule enlargement and neurite outgrowth by a calmodulin antagonism in N1E-115 cells. Recently, direct in vitro activation of protein kinase C by melatonin was described. Vimentin intermediate filaments are attached to microtubules and their organization depends on both microtubule distribution and phosphorylation of specific proteins. Protein kinase C is a serine threonine kinase which phosphorylates vimentin and through this mechanism causes intermediate filament disassembly. In this work the effects of melatonin on protein kinase C activation, content, and subcellular distribution were studied in N1E-115 cells. Also, melatonin effects on vimentin phosphorylation and subcellular distribution were explored. The results show that melatonin both activates and increases protein kinase C content in the membrane cytoskeletal fraction. Melatonin protein kinase C activation was followed by an increase in both vimentin phosphorylation and by vimentin subcellular redistribution. Moreover, staurosporine, a serine threonine kinase inhibitor, prevented increased vimentin phosphorylation elicited by melatonin. Similar effects to those caused by melatonin were obtained with the protein kinase C activator phorbol 12-myristate 13-acetate. Data support the idea that melatonin modulates vimentin organization through protein kinase C activation.
Asunto(s)
Filamentos Intermedios/efectos de los fármacos , Filamentos Intermedios/metabolismo , Melatonina/farmacología , Proteína Quinasa C/metabolismo , Vimentina/metabolismo , Animales , Línea Celular , Activación Enzimática/efectos de los fármacos , Ratones , Fosforilación , Estaurosporina/farmacología , Fracciones Subcelulares/enzimología , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Calmodulin (CaM) through activation of CaM-kinase II may be involved in the molecular mechanisms underlying the epileptogenic processes. Some evidence suggests that kindling responses change across the day-night cycle. In order to test if kindling stimulation modifies CaM content, we measured CaM concentrations in amygdala, hippocampus and hypothalamus obtained from control and kindled rats during light and darkness. Male Wistar rats (250-300 g), were injected i.p. with Pentylenetetrazol (PTZ) (35 mg/kg/24 h). Once chemical kindling was established, rats were sacrificed by decapitation at 10:30 a.m. and 01:30 a.m. The brains were obtained, and the amygdala, hippocampus and hypothalamus dissected. CaM content was measured in the cytosol and membrane fractions by radioimmunoassay. We found a significant increase in CaM content in cytosol and membrane fractions of both control and kindled rats during the dark phase. No significant differences in CaM concentrations were observed between control and experimental rats, whether during the light or the dark phase. The data suggest a well defined photoperiodic variation in CaM concentrations in limbic structures, despite the neuronal excitability produced by kindling. In addition, the observed CaM increases during the dark time may be related to a protective mechanism against enhanced sensitivity to seizures observed during the night.
Asunto(s)
Encéfalo/fisiología , Calmodulina/metabolismo , Ritmo Circadiano/fisiología , Excitación Neurológica/fisiología , Pentilenotetrazol/farmacología , Amígdala del Cerebelo/metabolismo , Animales , Encéfalo/efectos de los fármacos , Oscuridad , Hipocampo/metabolismo , Hipotálamo/metabolismo , Luz , Masculino , Especificidad de Órganos , Ratas , Ratas Wistar , Valores de ReferenciaRESUMEN
In this article, we review the data concerning melatonin interactions with calmodulin. The kinetics of melatonin-calmodulin binding suggest that the hormone modulates cell activity through intracellular binding to the protein at physiological concentration ranges. Melatonin interaction with calmodulin may allow the hormone to modulate rhythmically many cellular functions. Melatonin's effect on tubulin polymerization, and cytoskeletal changes in MDCK and N1E-115 cells cultured with melatonin, suggest that at low concentrations (10(-9) M) cytoskeletal effects are mediated by its antagonism to Ca2+-calmodulin. At higher concentrations (10(-5)M) non-specific binding of melatonin to tubulin occurs thus overcoming the specific melatonin antagonism to Ca2+-calmodulin. Since the structures of melatonin and calmodulin are phylogenetically well preserved, calmodulin-melatonin interaction probably represents a major mechanism for regulation and synchronization of cell physiology.
Asunto(s)
Calmodulina/metabolismo , Citoesqueleto/efectos de los fármacos , Melatonina/metabolismo , Animales , Calmodulina/antagonistas & inhibidores , Línea Celular , Citoesqueleto/metabolismo , Perros , Melatonina/farmacología , Ratones , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Transducción de Señal , Tubulina (Proteína)/metabolismo , Células Tumorales CultivadasRESUMEN
In vitro formation of a functional MDCK cell monolayer requires the dynamic participation of the cytoskeleton. Cell shape, contacts and polarity, as well as transepithelial electric resistance (TER), are actively modified during this differentiation process. We studied the distribution and rearrangement of cytokeratin, vimentin and actin filaments that occur in the monolayer concomitant with the synthesis and phosphorylation of these proteins. Cells cultured for short time in suspension, subconfluent or early confluent cells, showed striking differences in cell shape and arrangement of their cytoskeletal filaments. Subconfluent and early confluent cells synthesized proteins at high levels. In contrast, suspended cells maintained lower rates of protein synthesis. Labeled amino acid uptake was very similar in all these culture conditions. Gel electrophoresis analyses of the synthesized proteins showed increases in the synthesis of actin, vimentin and specific cytokeratins in subconfluent and early confluent cells. On the other hand a decrease in total protein, actin, vimentin and cytokeratin synthesis was observed in cells kept in suspension for 24 h or in 78-h or 174-h older confluent cultures. These cultures also showed a decrease in the uptake of labeled amino acid. Cytokeratin and vimentin phosphorylation rates were also modified during the in vitro formation of a monolayer. In suspended cells, cytokeratins were phosphorylated and three labeled isoelectric variants of the 40, 48 and 58K (K = 10(3) Mr) cytokeratins were present in intermediate filament extracts. In subconfluent and early confluent cells only two isoelectric phosphorylated variants of the 40, 48 and 58K cytokeratins were detected and vimentin was also phosphorylated.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas del Citoesqueleto/biosíntesis , Actinas/ultraestructura , Animales , Anticuerpos , Línea Celular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Proteínas del Citoesqueleto/ultraestructura , Electroforesis en Gel de Poliacrilamida , Filamentos Intermedios/ultraestructura , Metionina/metabolismo , Peso Molecular , Mapeo Peptídico , Fosforilación , Procesamiento Proteico-PostraduccionalRESUMEN
Despite the fact that many physiological and pharmacological actions of melatonin (MEL) have been described, its mechanism of action at the subcellular level remains unclear. It has been suggested that MEL has effects on cellular processes that involve microfilaments and microtubules. In the present study MEL effects on the cytoskeleton were evaluated in MDCK and N1E-115 cells in which the microfilaments have been shown to participate in cell morphology and dome formation (MDCK) and the microtubules in neurite outgrowths. After one day of culture with 10(-11)-10(-7) M MEL MDCK cells showed an increase in the number of elongated cells. After four days with the hormone, an increase in the incidence of MDCK cells contacting neighboring cells through long cytoplasmic elongations was observed. Actin antibody stain showed the appearance of thicker fluorescent fibres beneath the cell membrane and over the nucleus in the MEL treated cells. An increase in dome formation in confluent cells was also observed. In N1E-115 cells MEL (10(-13)-10(-5) M) induced an increase in cell with neurite processes. Neurite outgrowth is clearly seen at 24 h after plating. MEL-treated cells grow in clusters with neurites forming intricate networks. Antitubulin antibody stain showed long fluorescent neurites in the N1E-115 MEL-treated cells. A decrease in N1E-115 neurite formation was observed with either serotonin or 6-hydroxymelatonin (6OH-MEL). However, the number of MDCK cells with cytoplasmic elongations was decreased only after 6OH-MEL. We conclude that MEL action at the cellular level involves a modification of the cytoskeletal organization.
Asunto(s)
Citoesqueleto/efectos de los fármacos , Melatonina/farmacología , Citoesqueleto de Actina/efectos de los fármacos , Animales , Axones/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Microtúbulos/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Melatonin may play a key role in cytoskeletal rearrangements through its calmodulin antagonism. In the present work, we tested this hypothesis by studying melatonin effects on both microtubule polymerization in vitro and cytoskeletons in situ. Microtubule assembly is a dynamic process inhibited by Ca2+/calmodulin. Calmodulin antagonists prevent the inhibition by binding to Ca(2+)-activated calmodulin, thus causing microtubule enlargement. In the presence of calmodulin (5 microM) and CaCl2 (1 mM), polymerization at equilibrium was inhibited by 40%. Complete reversal of the Ca2+/calmodulin effect on microtubules was observed with 10(-9) M melatonin or with 10(-5) M trifluoperazine or 1 microgram/ml of compound 48/80. In the absence of Ca2+/calmodulin, melatonin at 10(-5) M inhibited tubulin polymerization like 10(-4) M trifluoperazine does. Melatonin effects on microtubule assembly at both nanomolar and micromolar ranges were corroborated in cytoskeletons in situ. Therefore, it is suggested that at a low concentration (10(-9) M), cytoskeletal melatonin effects are mediated by its antagonism to Ca2+/calmodulin. At a higher concentration (10(-5) M), non-specific binding of melatonin to tubulin occurs, thus overcoming the melatonin antagonism to Ca2+/calmodulin. The results support the hypothesis that under physiological conditions, melatonin synchronizes different body rhythms through cytoskeletal rearrangements mediated by its calmodulin antagonism.
Asunto(s)
Calmodulina/antagonistas & inhibidores , Melatonina/farmacología , Microtúbulos/efectos de los fármacos , Animales , Calcio/metabolismo , Calcio/farmacología , Calmodulina/metabolismo , Calmodulina/farmacología , Relación Dosis-Respuesta a Droga , Melatonina/fisiología , Ratones , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Neuroblastoma , Trifluoperazina/farmacología , Tubulina (Proteína)/metabolismo , Células Tumorales Cultivadas , p-Metoxi-N-metilfenetilamina/farmacologíaRESUMEN
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.
Asunto(s)
Entamoeba histolytica/enzimología , Fibronectinas/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Actinas/metabolismo , Alcaloides/farmacología , Animales , Adhesión Celular , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Entamoeba histolytica/efectos de los fármacos , Activación Enzimática , Isoquinolinas/farmacología , Fosforilación , Piperazinas/farmacología , Proteína Quinasa C/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Estaurosporina , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
In this paper we describe the interaction of melatonin with calmodulin in MDCK cells. The double staining immunofluorescent method showed that calmodulin in control MDCK cells appeared as fluorescent spots at the cell periphery. In contrast, MDCK cells cultured with 10(-9) M melatonin for 6, 12, 24 hr or for 4 days showed that calmodulin fluorescent spots were distributed throughout the cytoplasm as well as in the nucleus. These calmodulin rearrangements were reversed 6 hr after melatonin withdrawal. Moreover, calmodulin radioimmunoassay showed that in 10(-9) M melatonin-treated cells, membrane-bound calmodulin content was increased by 78% whereas cytosolic calmodulin decreased by 60%. Simultaneous labeling of MDCK cells with specific antibodies against calmodulin and melatonin showed that not only does the indole enter the cell, but that it has the same subcellular distribution as calmodulin. Besides the early responses induced by the melatonin antagonism to calmodulin, the results suggest that the indole may also induce long-term cellular responses by changing calmodulin concentrations in specific cellular compartments.
Asunto(s)
Calmodulina/metabolismo , Riñón/metabolismo , Melatonina/farmacología , Animales , Células Cultivadas , Citosol/metabolismo , Perros , Técnica del Anticuerpo Fluorescente Indirecta , Immunoblotting , Riñón/citología , Riñón/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Radioinmunoensayo , Fracciones Subcelulares/metabolismoRESUMEN
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
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.