RESUMO
Consumption of caffeine, a non-selective adenosine A2A receptor (A2AR) antagonist, reduces the risk of developing Alzheimer's disease (AD) in humans and mitigates both amyloid and Tau burden in transgenic mouse models. However, the impact of selective A2AR blockade on the progressive development of AD-related lesions and associated memory impairments has not been investigated. In the present study, we removed the gene encoding A2AR from THY-Tau22 mice and analysed the subsequent effects on both pathological (Tau phosphorylation and aggregation, neuro-inflammation) and functional impairments (spatial learning and memory, hippocampal plasticity, neurotransmitter profile). We found that deleting A2ARs protect from Tau pathology-induced deficits in terms of spatial memory and hippocampal long-term depression. These effects were concomitant with a normalization of the hippocampal glutamate/gamma-amino butyric acid ratio, together with a global reduction in neuro-inflammatory markers and a decrease in Tau hyperphosphorylation. Additionally, oral therapy using a specific A2AR antagonist (MSX-3) significantly improved memory and reduced Tau hyperphosphorylation in THY-Tau22 mice. By showing that A2AR genetic or pharmacological blockade improves the pathological phenotype in a Tau transgenic mouse model, the present data highlight A2A receptors as important molecular targets to consider against AD and Tauopathies.
Assuntos
Transtornos Cognitivos/fisiopatologia , Hipocampo/fisiopatologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptor A2A de Adenosina/metabolismo , Tauopatias/fisiopatologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Animais , Transtornos Cognitivos/tratamento farmacológico , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Hipocampo/efeitos dos fármacos , Humanos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Camundongos Transgênicos , Fosforilação , RNA Mensageiro/metabolismo , Receptor A2A de Adenosina/genética , Tauopatias/tratamento farmacológico , Técnicas de Cultura de Tecidos , Xantinas/farmacologia , Ácido gama-Aminobutírico/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal lung disease of which the origin and development mechanisms remain unknown. The few available pharmacological treatments can only slow the progression of the disease. The development of curative treatments is hampered by the absence of experimental models that can mimic the specific pathophysiological mechanisms of IPF. The aim of this mini-review is to provide an overview of the most commonly used experimental animal models in the study of IPF and to underline the urgent need to seek out new, more satisfactory models.
Assuntos
Fibrose Pulmonar Idiopática , Animais , Humanos , Fibrose Pulmonar Idiopática/diagnóstico , Fibrose Pulmonar Idiopática/terapiaRESUMO
Neuroendocrine differentiation (NED) has been implicated in prostate cancer progression and hormone-therapy failure. Neuroendocrine cells are non-proliferating and escape apoptotic cell death, although their origin and the causes of their apoptotic resistance have as yet been poorly elucidated. This study demonstrates a new mechanism involved in controlling NED. We report that epidermal growth factor (5-50 ng/ml) promotes neuroendocrine-like differentiation of androgen-independent DU145 prostate cancer cells. This differentiation is associated with an increase in the expression of Neuron Specific Enolase (NSE) and a reduction in cell proliferation and is blocked by inhibiting tyrosine kinase activity with genistein and with compound 56 (C56). An increase in the cAMP level, using dibutryl cAMP (db-cAMP) (1 mM) and isobutylmethylxanthine (100 microM), does not promote NED by itself, but does increase the effect of EGF on NED. In addition, EGF-induced NED protects cells from apoptosis induced with thapsigargin (1 microM) by reducing the thapsigargin-induced cytosolic calcium overload. In order to describe how EGF-induced NED protects cells against thapigargin-induced calcium overload we investigated the spatiotemporal calcium signalling linked to apoptosis. By using thapsigargin in various conditions on DU145 cells and using micro-fluorimetric calcium measurements, we show that depletion of intracellular calcium store induces apoptosis and that the amplitude and duration of the capacitive calcium entry are two apoptosis-modulating parameters. We show that protection against thapsigargin-induced apoptosis conferred by NED is achieved by reducing the amount and the speed of calcium that can be released from calcium pools, as well as modulating the amplitude of the subsequent calcium entry.
Assuntos
Androgênios/metabolismo , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Neoplasias Hormônio-Dependentes/patologia , Sistemas Neurossecretores/efeitos dos fármacos , Neoplasias da Próstata/patologia , Antineoplásicos/farmacologia , Cálcio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Humanos , Masculino , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias Hormônio-Dependentes/metabolismo , Fosfopiruvato Hidratase/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Tapsigargina/farmacologiaRESUMO
In normal medium supplemented with 10 mM tetraethylammonium chloride (TEACl), membrane depolarizations of immature Rana esculenta oocytes elicited an oscillatory outward current associated with a voltage-dependent H+ current (IH+). The voltage threshold of these oscillations was 22 +/- 5 (n = 10). The oscillations were blocked by intracellular injection of ethylene glycol-O,O'-bis-(2-acetaminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), by application of 1 mM of 4-acetamido-4'-isocyanatostilbene-2,2'-disulfonic acid (SITS), by caffeine (1 mM), and by the intracellular injection of heparin, suggesting that they arose from calcium release from inositol trisphosphate (InsP3)-sensitive stores, monitored by a calcium-dependent chloride current (IClCa2+). The oscillations were independent of the external calcium concentration, and the depolarizations did not affect the InsP3 level. Ni2+, a IH+ inhibitor, blocked the oscillations. Extracellular alkalinization, which lowered the voltage threshold of IH+ and increased its amplitude, also lowered the voltage threshold of the oscillations and increased their amplitude, whereas extracellular acidification produced opposite effects. We suggest that the oscillations are linked to activation of IH+ through a pH-dependent sensitization of InsP3 receptors.
Assuntos
Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Prótons , Animais , Cloretos/metabolismo , Condutividade Elétrica , Eletrofisiologia , Oócitos/fisiologia , Rana esculenta/fisiologiaRESUMO
Voltage clamp technique was used to study macroscopic ionic currents in Rana esculenta oocytes. Depolarization steps led to the activation of a single type of outward current (Iout) when contaminant potassium and calcium-dependent chloride currents were pharmacologically inhibited. The voltage threshold of Iout activation was 10 mV and this current, which did not inactivate, presented a deactivation the time constant of 73 +/- 21 msec (n = 26) corresponding to a membrane voltage of -60 mV. Its reversal potential (Erev) was dependent on the magnitude of the depolarization and also on pulse duration. These changes in Erev were thought to reflect intracellular ion depletion occurring during activation of the remaining outward current. Furthermore, the activation threshold of Iout was clearly affected by modifications in extracellular and intracellular H+ concentrations. Indeed, intracellular alkalinization (evoked by external application of ammonium chloride) or extracellular acidification induced a rightward shift in the activation threshold while intracellular acidification (evoked by external application of sodium acetate) or extracellular alkalinization shifted this threshold toward a more negative value. Lastly, Iout was dramatically reduced by divalent cations such as Cd2+, Ni2+ or Zn2+ and was strongly decreased by 4 Aminopyridine (4-AP), well-known H+ current antagonists already described in many cell types. Therefore, it was suggested that the outward current was prominently carried by H+ ions, which may play a key role in the regulation of intracellular pH and subsequent pH dependent processes in Rana oocyte.
Assuntos
Oócitos/metabolismo , Prótons , 4-Aminopiridina/farmacologia , Animais , Cátions Bivalentes/farmacologia , Cloretos/farmacologia , Feminino , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana , Metais/farmacologia , Oócitos/efeitos dos fármacos , Potássio/farmacologia , Rana esculenta , Sódio/farmacologiaRESUMO
When the capping of membrane immunoglobulin on spleen B cells from normal C57Bl/6 mice (B6) is taken as reference, a faster capping rate is found for cells of age-matched B6 mice which are congenic at the lymphoproliferation (lpr) or nude (nu) loci. Though both congenic strains can be characterized by an abnormal T-lineage cell content, the nature of the abnormality itself is very different since B6 nudes lack thymus-processed/influenced lymphocytes whereas B6 mice with the lpr phenotype suffer from an invasion of all lymphoid organs with cells of a particular T-cell subset. Moreover, the more "normal" capping rate of B cells from the double congenic B6 mice (nu/nu, lpr/lpr) is intriguing. Since other mice homozygous at the lpr locus (MRL-1) or at the nu locus (BALB/c nude) also cap faster than their congenic controls (MRL-n and BALB/c, respectively), the observed effects do not appear to depend on a peculiarity of the B6 genetic background. If the faster capping of B cells of nu congenic and of lpr congenic mice had a common origin, it might be that T cells would control in some way the mobility of B-cell membrane immunoglobulins: both congenic mice have in their spleen a very low proportion of mature T cells together with a very high proportion of prethymic/thymic immature T-cell types, either of which might affect B-cell behavioral responses to membrane immunoglobulin clustering.
Assuntos
Linfócitos B/imunologia , Genes , Capeamento Imunológico , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Ciclosporinas/farmacologia , Feminino , Homozigoto , Capeamento Imunológico/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Baço/imunologiaRESUMO
The immunomodulatory molecule cyclosporine was found to cause an early acceleration of the capping of mouse B-cell membrane immunoglobulin. This was most marked when the capping process was slightly retarded by keeping the cells at 32 degrees. This is a sign that the drug can cause B-cell membrane alterations. At lower drug doses they might not be as easily detected, but are nevertheless sufficient to inhibit the activation of some B cells by membrane immunoglobulin clustering.
Assuntos
Linfócitos B/imunologia , Ciclosporinas/farmacologia , Capeamento Imunológico/efeitos dos fármacos , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Células Cultivadas , Feminino , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos B/análise , TemperaturaRESUMO
The expression in Xenopus oocytes of the human voltage-dependent Ca2+ channel (VDCC) beta 2 subunit subtype (h beta 2) enhances the endogenous Ca2+ channel activity. By using the native Ca(2+)-dependent chloride conductance to monitor fast intracellular Ca2+ variations, we point out that the beta-enhanced Ca2+ entry (T1 component) is currently associated with a second delayed elevation of internal Ca2+ (T2 component). Further experiments show that this additional component absolutely requires Ca2+ entry through the beta-modulated channels although it directly derives from a Ca2+ release from intracellular inositol (1,4,5)-trisphosphate (InsP3)-sensitive stores. Finally, our study demonstrates that InsP3-evoked response in oocytes is dramatically modified since it gains a new shape of voltage dependency directly derived from the beta-modified Ca2+ influx. The main conclusion is that the spatiotemporal pattern of InsP3-dependent Ca2+ release may be closely influenced by the intrinsic characteristics of working VDCCs.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Cádmio/farmacologia , Canais de Cálcio/biossíntese , Cloretos/metabolismo , Primers do DNA , Feminino , Heparina/farmacologia , Humanos , Substâncias Macromoleculares , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Transdução de Sinais/efeitos dos fármacos , Xenopus laevisRESUMO
The present study demonstrates for the first time that intracellular calcium-ATPases and calcium pool content are closely associated with prostate cancer LNCaP cell growth. Cell growth was modulated by changing the amount of epidermal growth factor, serum, and androgene in culture media. Using the microspectrofluorimetric method with Fura-2 and Mag Fura-2 as probes, we show that in these cells, the growth rate is correlated with intracellular calcium pool content. Indeed, an increased growth rate is correlated with an increase in the calcium pool filling state, whereas growth-inhibited cells show a reduced calcium pool load. Using Western blotting and immunocytochemistry, we show that endoplasmic reticulum calcium pump expression is closely linked to LNCaP cell growth, and are a common target of physiological stimuli that control cell growth. Moreover, we clearly demonstrate that inhibition of these pumps, using thapsigargin, inhibits LNCaP cell growth and prevents growth factor from stimulating cell proliferation. Our results thus provide evidence for the essential role of functional endoplasmic reticulum calcium pumps and calcium pool in control of prostate cancer LNCaP cell growth, raising the prospect of new targets for the treatment of prostate cancer.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Retículo Endoplasmático/enzimologia , Neoplasias da Próstata/metabolismo , Retículo Sarcoplasmático/enzimologia , Western Blotting , ATPases Transportadoras de Cálcio/biossíntese , Divisão Celular , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/antagonistas & inibidores , Corantes Fluorescentes/farmacologia , Fura-2/farmacologia , Humanos , Imuno-Histoquímica , Membranas Intracelulares/metabolismo , Masculino , Microscopia de Fluorescência , Microssomos/metabolismo , Ligação Proteica , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Espectrofotometria , Tapsigargina/farmacologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
1. In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+-ATPase inhibitor thapsigargin (TG), in androgen-sensitive human prostate cancer LNCaP cells. 2. Exposure of fura-2-loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store-operated Ca2+ channels (SOCs). 3. For the first time we have identified and characterized the SOC-mediated membrane current (Istore) in prostate cells using whole-cell, cell-attached, and perforated patch-clamp techniques, combined with fura-2 microspectrofluorimetric and Ca2+-imaging measurements. 4. Istore in LNCaP cells lacked voltage-dependent gating and displayed an inwardly rectifying current-voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 +/- 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5-3 mM) and La3+ (1 microM). 5. Treatment of LNCaP cells with TG (0.1 microM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG-induced apoptosis. 6. The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA-AM). 7. These results indicate that in androgen-sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store-activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death.