RESUMEN
Growing evidence suggests a close relationship between Alzheimer's Disease (AD) and cerebral hypoxia. Astrocytes play a key role in brain homeostasis and disease states, while some of the earliest changes in AD occur in astrocytes. We have therefore investigated whether mutations associated with AD increase astrocyte vulnerability to ischemia. Two astroglioma cell lines derived from APPSWE /PS1A246E (APP, amyloid precursor protein; PS1, presenilin 1) transgenic mice and controls from normal mice were subjected to oxygen and glucose deprivation (OGD), an in vitro model of ischemia. Cell death was increased in the APPSWE /PS1A246E line compared to the control. Increasing extracellular calcium concentration ([Ca2+ ]) exacerbated cell death in the mutant but not in the control cells. In order to explore cellular Ca2+ homeostasis, the cells were challenged with ATP or thapsigargin and [Ca2+ ] was measured by fluorescence microscopy. Changes in cytosolic Ca2+ concentration ([Ca2+ ]c ) were potentiated in the APPSWE /PS1A246E transgenic line. Mitochondrial function was also altered in the APPSWE /PS1A246E astroglioma cells; mitochondrial membrane potential and production of reactive oxygen species were increased, while mitochondrial basal respiratory rate and ATP production were decreased compared to control astroglioma cells. These results suggest that AD mutations in astrocytes make them more sensitive to ischemia; Ca2+ dysregulation and mitochondrial dysfunction may contribute to this increased vulnerability. Our results also highlight the role of astrocyte dyshomeostasis in the pathophysiology of neurodegenerative brain disorders.
Asunto(s)
Enfermedad de Alzheimer , Astrocitos , Isquemia Encefálica , Calcio/metabolismo , Mitocondrias/patología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/genética , Animales , Astrocitos/metabolismo , Astrocitos/patología , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Línea Celular , Glucosa/deficiencia , Ratones , Ratones Transgénicos , Mutación , Oxígeno , Presenilina-1/genéticaRESUMEN
Therapeutic options for amyotrophic lateral sclerosis (ALS) are scarce and controversial. Although the aetiology of neuronal vulnerability is unknown, growing evidence supports a complex network in which multiple toxicity pathways, rather than a single mechanism, are involved in the pathogenesis of ALS. However, most cellular models only explain single pathogenic mechanisms. The present study proposes the two main cytotoxic mechanisms: (1) veratridine (VTD), which induced Na+ and Ca2+ overload; and (2) the TARD DNA-binding protein 43 (TDP-43) in NSC-34 cell line as an in vitro model of ALS. The study was carried out by MTT as an indirect measurement of cell viability and by flow cytometry to determine cell death stages. The impact of Ca2+ overload combined with TDP-43 overexpression increased early apoptosis of NSC-34 cells. Furthermore, we found that ITH33/IQM9.21 (ITH33) exerted a neuroprotective effect in this model by reducing activation of the apoptotic pathway. Therefore, treatment with VTD in TDP-43 overexpressing NSC-34 cells is a good in vitro ALS model that makes it possible to test new neuroprotective compounds such as ITH33.
Asunto(s)
Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Benzamidas/farmacología , Calcio/metabolismo , Proteínas de Unión al ADN/metabolismo , Glutamatos/farmacología , Fármacos Neuroprotectores/farmacología , Sodio/metabolismo , Veratridina/toxicidad , Animales , Apoptosis , Cationes Bivalentes , Cationes Monovalentes , Línea Celular , Supervivencia Celular , Humanos , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Neuronas Motoras/patologíaRESUMEN
Alternatives for the treatment of amyotrophic lateral sclerosis (ALS) are scarce and controversial. The etiology of neuronal vulnerability in ALS is being studied in motor neuron-like NSC-34 cells to determine the underlying mechanisms leading to selective loss of motor neurons. One such mechanism is associated with mitochondrial oxidative stress, Ca(2+) overload, and low expression of Ca(2+)-buffering proteins. Therefore, in order to elicit neuronal death in ALS, NSC-34 cells were exposed to the following cytotoxic agents: (1) a mixture of oligomycin 10 µM and rotenone 30 µM (O/R), or (2) phenylarsine oxide 1 µM (PAO) (to mimic excess free radical production during mitochondrial dysfunction), and (3) veratridine 100 µM (VTD) (to induce overload of Na(+) and Ca(2+) and to alter distribution of Ca(2+)-buffering proteins [parvalbumin and calbindin-D28k]). Thus, the aim of the study was to test the novel neuroprotective compound ITH33/IQM9.21 (ITH33) and to compare it with riluzole on in vitro models of neurotoxicity. Cell viability measured with MTT showed that only ITH33 protected against O/R at 3 µM and PAO at 10 µM, but not riluzole. ITH33 and riluzole were neuroprotective against VTD, blocked the maximum peak and the number of [Ca(2+)]c oscillations per cell, and restored the effect on parvalbumin. However, only riluzole reversed the effect on calbindin-D28k levels. Therefore, ITH33 was neuroprotective against oxidative stress and Na(+)/Ca(2+) overload, both of which are involved in ALS.
Asunto(s)
Benzamidas/farmacología , Glutamatos/farmacología , Neuronas Motoras/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/efectos de los fármacos , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Arsenicales , Calbindina 1/metabolismo , Calcio/metabolismo , Calcio/toxicidad , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Evaluación Preclínica de Medicamentos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas Motoras/patología , Oligomicinas/toxicidad , Estrés Oxidativo/fisiología , Parvalbúminas/metabolismo , Riluzol/farmacología , Rotenona/toxicidad , Sodio/metabolismo , Sodio/toxicidad , Veratridina/toxicidadRESUMEN
The mitochondrial Na(+)/Ca(2+) exchanger plays an important role in the control of cytosolic Ca(2+) cycling in excitable cells, essential for the regulation of a plethora of Ca(2+)-dependent physio-pathological events, such as apoptosis in the presence of a Ca(2+) overload. There are very few pharmacological tools available to study both physiological and pathological implications of the mitochondrial Na(+)/Ca(2+) exchanger, where the benzothiazepine CGP37157 is the best-known ligand, used since the 1980s. However, it is not an efficient blocker and lacks of selectivity, as also blocks several other cellular Ca(2+) transporters. Moreover, CGP37157 is a very lipophilic drug, showing very poor water solubility, what has hindered its therapeutic use. Attempting to improve its pharmacokinetic profile as well as its potency and selectivity, we herein describe the synthesis of new CGP37157 analogs, where the benzene-fused ring has been replaced by a pyridine. On top of a better water solubility and lower log P value, some of these new pyridothiazepine derivatives also presented a higher capacity to regulate the mitochondrial Ca(2+) clearance, while keeping the neuroprotective properties presented in the head compound CGP37157.
Asunto(s)
Mitocondrias/efectos de los fármacos , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Tiazepinas/química , Tiazepinas/farmacología , Animales , Calcio/metabolismo , Bovinos , Línea Celular , Línea Celular Tumoral , Células HeLa , Hipocampo/efectos de los fármacos , Humanos , Mitocondrias/metabolismo , Fármacos Neuroprotectores/farmacocinética , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacología , Ratas , Intercambiador de Sodio-Calcio/metabolismo , Solubilidad , Tiazepinas/farmacocinéticaRESUMEN
Mitochondria regulate cellular Ca(2+) oscillations, taking up Ca(2+) through its uniporter and releasing it through the mitochondrial sodium/calcium exchanger. The role of mitochondria in the regulation of Ca(2+) cycle has received much attention recently, as it is a central stage in neuronal survival and death processes. Over the last decades, the 4,1-benzothiazepine CGP37157 has been the only available blocker of the mitochondrial sodium/calcium exchanger, although it targets several other calcium transporters. We report the synthesis of 4,1-benzothiazepine derivatives with the goal of enhancing mitochondrial sodium/calcium exchanger blockade and selectivity, and the evaluation of their cytoprotective effect. The compound 4c presented an interesting neuroprotective profile in addition to an important blockade of the mitochondrial sodium/calcium exchanger. The use of this benzothiazepine could help to understand the physiological functions of the mitochondrial sodium/calcium exchanger. In addition, we hypothesize that a moderate blockade of the mitochondrial sodium/calcium exchanger would provide enhanced neuroprotection in neurons.
Asunto(s)
Enfermedades Neurodegenerativas/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Tiazepinas/farmacología , Animales , Calcio/metabolismo , Bovinos , Muerte Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cromafines/efectos de los fármacos , Células Cromafines/fisiología , Citoprotección , Evaluación Preclínica de Medicamentos , Células HeLa , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estructura Molecular , Fármacos Neuroprotectores/síntesis química , Fármacos Neuroprotectores/química , Sodio/metabolismo , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/metabolismo , Tiazepinas/síntesis química , Tiazepinas/químicaRESUMEN
CALHM1 is a Ca(2+) channel discovered in 2008, which plays a key role in the neuronal electrical activity, among other functions. However, there are no known efficient blockers able to modulate its Ca(2+) handling ability. We herein describe that benzothiazepine CGP37157 and its newly synthesized analogue ITH12575 reduced Ca(2+) influx through CALHM1 at low micromolar concentrations. These results could serve as a starting point for the development of more selective CALHM1 ligands using CGP37157 as a hit compound, which would help to study the physiological role of CALHM1 in the control of [Ca(2+)]cyt in excitable cells, as well as its implication in CNS diseases.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Calcio/metabolismo , Clonazepam/análogos & derivados , Glicoproteínas de Membrana/antagonistas & inhibidores , Tiazepinas/farmacología , Bloqueadores de los Canales de Calcio/síntesis química , Bloqueadores de los Canales de Calcio/química , Canales de Calcio/genética , Canales de Calcio/metabolismo , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Cationes/metabolismo , Clonazepam/química , Clonazepam/farmacología , Cobalto/metabolismo , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Indoles/farmacología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Polimorfismo Genético , Ionóforos de Protónes/farmacología , Tiazepinas/síntesis química , Tiazepinas/química , TransfecciónRESUMEN
Positive allosteric modulation of α7 isoform of nicotinic acetylcholine receptors (α7-nAChRs) is emerging as a promising therapeutic approach for central nervous system disorders such as schizophrenia or Alzheimer's disease. However, its effect on Ca(2+) signaling and cell viability remains controversial. This study focuses on how the type II positive allosteric modulator (PAM II) PNU120596 affects intracellular Ca(2+) signaling and cell viability. We used human SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH). We monitored cytoplasmic and endoplasmic reticulum (ER) Ca(2+) with Fura-2 and the genetically encoded cameleon targeting the ER, respectively. Nicotinic inward currents were measured using patch-clamp techniques. Viability was assessed using methylthiazolyl blue tetrazolium bromide or propidium iodide staining. We observed that in the presence of a nicotinic agonist, PNU120596 (i) reduced viability of α7-SH but not of C-SH cells; (ii) significantly increased inward nicotinic currents and cytosolic Ca(2+) concentration; (iii) released Ca(2+) from the ER by a Ca(2+) -induced Ca(2+) release mechanism only in α7-SH cells; (iv) was cytotoxic in rat organotypic hippocampal slice cultures; and, lastly, all these effects were prevented by selective blockade of α7-nAChRs, ryanodine receptors, or IP3 receptors. In conclusion, positive allosteric modulation of α7-nAChRs with the PAM II PNU120596 can lead to dysregulation of ER Ca(2+) , overloading of intracellular Ca(2+) , and neuronal cell death. This study focuses on how the type II positive allosteric modulator PNU120596 (PAM II PNU12) affects intracellular Ca(2+) signaling and cell viability. Using SH-SY5Y neuroblastoma cells overexpressing α7-nAChRs (α7-SH) and their control (C-SH), we find that PAM of α7-nAChRs with PNU120596: (i) increases inward calcium current (ICa ) and cytosolic Ca(2+) concentration ([Ca(2+) ]cyt ); (ii) releases Ca(2+) from the ER ([Ca(2+) ]ER ) by a Ca(2+) -induced Ca(2+) release mechanism; and (iv) reduces cell viability. These findings were corroborated in rat hippocampal organotypic cultures. [Ca(2+) ]cyt , cytosolic Ca(2+) concentration; [Ca(2+) ]ER , endoplasmic reticulum Ca(2+) concentration; α7 nAChR, α7 isoform of nicotinic acetylcholine receptors; α7-SH, SH-SY5Y stably overexpressing α7 nAChRs cells; C-SH, control SH-SY5Y cells; Nic, nicotine; PNU12, PNU120596.
Asunto(s)
Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Receptor Nicotínico de Acetilcolina alfa 7/fisiología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Animales , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Humanos , Isoxazoles/farmacología , Masculino , Compuestos de Fenilurea/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The steroid Na(+)/K(+) ATPase (NKA) blocker ouabain has been shown to exhibit pro-apoptotic effects in various cell systems; however, the mechanism involved in those effects is unclear. Here, we have demonstrated that incubation of HeLa cells during 24h with nanomolar concentrations of ouabain or digoxin causes apoptotic death of 30-50% of the cells. Ouabain caused the activation of caspases-3/7 and -9; however, caspase-8 was unaffected. The fact that compound Z-LEHD-FMK reduced both apoptosis and caspase-9 activation elicited by ouabain, suggest a mitochondrially-mediated pathway. This was strengthened by the fact that ouabain caused ATP depletion and the release of mitochondrial cytochrome c into the cytosol. Furthermore, upon ouabain treatment mitochondrial disruption and redistribution into the cytosol were observed. A mitochondrial site of action for ouabain was further corroborated by tight co-localisation of fluorescent ouabain with mitochondria. Finally, in ouabain-treated cells the histamine-elicited elevation of cytosolic Ca(2+) concentration ([Ca(2+)]c) suggests an additional effect on the endoplasmic reticulum (ER) leading to Ca(2+) store depletion. We conclude that fluorescent ouabain is taken up and tightly co-localises with mitochondria of HeLa cells. This indicates that apoptosis may be triggered by a direct action of ouabain on mitochondria.
Asunto(s)
Apoptosis/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Ouabaína/farmacología , Transporte Biológico , Biomarcadores/metabolismo , Calcio/metabolismo , Caspasa 9/metabolismo , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células HeLa , Histamina/farmacología , Humanos , Mitocondrias/metabolismo , Oligopéptidos/farmacología , Factores de TiempoRESUMEN
In recent years, the antitumoral activity of antimicrobial peptides (AMPs) has been the goal of many research studies. Among AMPs, gomesin (Gm) displays antitumor activity by unknown mechanisms. Herein, we studied the cytotoxicity of Gm in the Chinese hamster ovary (CHO) cell line. Furthermore, we investigated the temporal ordering of organelle changes and the dynamics of Ca(2+) signaling during Gm-induced cell death. The results indicated that Gm binds to the plasma membrane and rapidly translocates into the cytoplasm. Moreover, 20 µM Gm increases the cytosolic Ca(2+) and induces membrane permeabilization after 30 min of treatment. Direct Ca(2+) measurements in CHO cells transfected with the genetically encoded D1-cameleon to the endoplasmic reticulum (ER) revealed that Gm induces ER Ca(2+) depletion, which in turn resulted in oscillatory mitochondrial Ca(2+) signal, as measured in cells expressing the genetically encoded probe to the mitochondrial matrix (mit)Pericam. This leads to mitochondria disruption, loss of mitochondrial membrane potential and increased reactive oxygen species prior to membrane permeabilization. Gm-induced membrane permeabilization by a Ca(2+)-dependent pathway involving Gm translocation into the cell, ER Ca(2+) depletion and disruption, mitochondrial Ca(2+) overload and oxidative stress.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Animales , Células CHO , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The augmentation of neurotransmitter and hormone release produced by ouabain inhibition of plasmalemmal Na+/K+-ATPase (NKA) is well established. However, the mechanism underlying this action is still controversial. Here we have shown that in bovine adrenal chromaffin cells ouabain diminished the mobility of chromaffin vesicles, an indication of greater number of docked vesicles at subplasmalemmal exocytotic sites. On the other hand, ouabain augmented the number of vesicles undergoing exocytosis in response to a K+ pulse, rather than the quantal size of single vesicles. Furthermore, ouabain produced a tiny and slow Ca2+ release from the endoplasmic reticulum (ER) and gradually augmented the transient elevations of the cytosolic Ca2+ concentrations ([Ca2+]c) triggered by K+ pulses. These effects were paralleled by gradual increments of the transient catecholamine release responses triggered by sequential K+ pulses applied to chromaffin cell populations treated with ouabain. Both, the increases of K+-elicited [Ca2+]c and secretion in ouabain-treated cells were blocked by thapsigargin (THAPSI), 2-aminoethoxydiphenyl borate (2-APB) and caffeine. These results are compatible with the view that ouabain may enhance the ER Ca2+ load and facilitate the Ca2+-induced-Ca2+ release (CICR) component of the [Ca2+]c signal generated during K+ depolarisation. This could explain the potentiating effects of ouabain on exocytosis.
Asunto(s)
Glándulas Suprarrenales/efectos de los fármacos , Calcio/metabolismo , Células Cromafines/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Exocitosis/efectos de los fármacos , Ouabaína/farmacología , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/fisiología , Animales , Compuestos de Boro/farmacología , Cafeína/farmacología , Catecolaminas/metabolismo , Bovinos , Células Cromafines/metabolismo , Citosol/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Inhibidores Enzimáticos/farmacología , Potasio/farmacología , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Tapsigargina/farmacologíaRESUMEN
The novel Ca(2+) channel CALHM1 (Calcium Homeostasis Modulator 1) generates cytosolic Ca(2+) transients ([Ca(2+)](c)) that regulate the production of amyloid beta (Abeta). Its mutated channel P86L-CALHM1 has been associated to Alzheimer's disease (AD). Using cytosolic- and mitochondrial-targeted aequorins, we have investigated here whether mitochondria sense with similar or different kinetics the Ca(2+) entering into Hela cells and the Ca(2+) released from the endoplasmic reticulum (ER), in control and in cells transfected with CALHM1 and P86L-CALHM1. We have shown that mitochondria sense Ca(2+) entry in the three cell types; however, the [Ca(2+)](c) and mitochondrial Ca(2+) transients [Ca(2+)](m) had substantially slower kinetics in cells expressing P86L-CALHM1. Mitochondria also sensed the ER Ca(2+) released by histamine, but in CALHM1 and P86L-CALHM1 cells the kinetics was faster than that of control cells. Data are compatible with the idea that mutated CALHM1 may cause mitochondrial Ca(2+) overload, suggesting how these cells may become more vulnerable to apoptotic stimuli.