RESUMEN
Abstract The prolonged entry of large amounts of calcium into the mitochondria through the mitochondrial calcium uniporter complex (MCUC) may cause the permeability transition pore (mPTP) to open, which contributes to the pathogenesis of several diseases. Tissue-specific differences in mPTP opening due to variable expression of MCUC components may contribute to disease outcomes. We designed this study to determine differential mPTP opening in mitochondria isolated from different regions of mouse brain and kidney and to compare it with the expression of MCUC components. mPTP opening was measured using mitochondria isolated from the left/right brain hemispheres (LH/RH, respectively) and from kidney cortex/medulla, while the expression level of MCUC components was assessed from total cellular RNA. Interestingly, LH mitochondria showed less calcium-induced mPTP opening as compared to RH mitochondria at two different calcium concentrations. Conversely, mPTP opening was similar in the renal cortex and renal medulla mitochondria. However, the kidney mitochondria demonstrated bigger and faster mPTP opening as compared to the brain mitochondria. Furthermore, asymmetric mPTP opening in the LH and RH mitochondria was not associated with the expression of MCUC components. In brief, this study demonstrates thus far unreported asymmetric mPTP opening in mouse brain hemispheres that is not associated with the mRNA levels of MCUC components.
Asunto(s)
Animales , Masculino , Femenino , Ratones , Encéfalo , Calcio/agonistas , Cerebro/anomalías , Poro de Transición de la Permeabilidad Mitocondrial/análisis , Ratones , Mitocondrias , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/efectos adversos , Corteza RenalRESUMEN
Abstract Ligustrazine is widely used for the treatment of cardiovascular diseases in traditional Chinese medication. It has been reported that Ligustrazine decreases the concentration of intracellular calcium ions (Ca2+); however, the underlying mechanism remains unknown. In the present study, the effect of Ligustrazine on adenosine diphosphate (ADP)-induced platelet aggregation was evaluated using a turbidimetric approach. The changes in concentration of intracellular Ca2+ stimulated by ADP was measured using fluo-4, a fluorescent Ca2+ indicator dye. The mRNA expression of stromal interaction molecule l (STIM1) and Orai1, calcium sensor, was determined using real-time PCR. In addition, the protein expression of STIM1, Orai1, and serum/glucocorticoid-regulated protein kinase 1 (SGK1) was determined using Western blot analysis. The data demonstrated that Ligustrazine significantly suppressed platelet aggregation in a dose-dependent manner and reduced the concentration of intracellular Ca2+ triggered by ADP. Our data showed that Ligustrazine treatment inhibited the expression of STIM1 and Orai1 induced by ADP at both mRNA and protein levels, and suppressed the protein expression of SGK1. Taken together, our data indicated that Ligustrazine suppressed platelet aggregation by partly inhibiting the activities of calcium sensors, thereby suggesting that Ligustrazine may be a promising candidate for the treatment of platelet aggregation.
Asunto(s)
Animales , Masculino , Ratas , Proteínas Quinasas , Enfermedades Cardiovasculares/patología , Agregación Plaquetaria , Adenosina Difosfato/farmacología , Western Blotting/métodos , Calcio/agonistas , Pueblo Asiatico/clasificación , Moléculas de Interacción EstromalRESUMEN
Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal muscle suggests that co-administration of ß-adrenergic and calcium agonists would activate complementary signaling relevant to this exercise context. The phosphoproteome of cells treated with this combination reveals a surprising divergence in signaling from the individual treatments. Remarkably, only the combination treatment promotes multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-fibrotic secreted protein. Secretome analysis reveals that the combined treatments decrease secretion of SERPINE1 and other deleterious factors. This study provides a framework for dissecting phosphorylation-based signaling relevant to acute exercise.
Asunto(s)
Ejercicio Físico/fisiología , Músculo Esquelético/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinasas/metabolismo , Proteoma/metabolismo , Transducción de Señal/fisiología , Estrés Fisiológico/genética , Quinasas de la Proteína-Quinasa Activada por el AMP , Agonistas Adrenérgicos beta/metabolismo , Animales , Aripiprazol/metabolismo , Aripiprazol/farmacología , Calcio/agonistas , Calcio/metabolismo , Interacciones Farmacológicas , Humanos , Isoproterenol/metabolismo , Isoproterenol/farmacología , Espectrometría de Masas , Ratones , Fosfoproteínas/química , Fosforilación , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidor 1 de Activador Plasminogénico/metabolismo , Sistemas de Translocación de Proteínas/genética , Sistemas de Translocación de Proteínas/metabolismo , Proteoma/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ratas , Estrés Fisiológico/fisiología , Tapsigargina/metabolismo , Tapsigargina/farmacologíaRESUMEN
Plasma calcium (Ca2+) is maintained by amending the release of parathyroid hormone and through direct effects of the Ca2+ sensing receptor (CaSR) in the renal tubule. Combined, these mechanisms alter intestinal Ca2+ absorption by modulating 1,25-dihydroxy vitamin D3 production, bone resorption, and renal Ca2+ excretion. The CaSR is a therapeutic target in the treatment of secondary hyperparathyroidism and hypocalcemia a common complication of calcimimetic therapy. The CaSR is also expressed in intestinal epithelium, however, a direct role in regulating local intestinal Ca2+ absorption is unknown. Chronic CaSR activation decreased expression of genes involved in Ca2+ absorption. In Ussing chambers, increasing extracellular Ca2+ or basolateral application of the calcimimetic cinacalcet decreased net Ca2+ absorption across intestinal preparations acutely. Conversely, Ca2+ absorption increased with decreasing extracellular Ca2+ concentration. These responses were absent in mice expressing a non-functional TRPV6, TRPV6D541A. Cinacalcet also attenuated Ca2+ fluxes through TRPV6 in Xenopus oocytes when co-expressed with the CaSR. Moreover, the phospholipase C inhibitor, U73122, prevented cinacalcet-mediated inhibition of Ca2+ flux. These results reveal a regulatory pathway whereby activation of the CaSR in the basolateral membrane of the intestine directly attenuates local Ca2+ absorption via TRPV6 to prevent hypercalcemia and help explain how calcimimetics induce hypocalcemia.
Asunto(s)
Calcimiméticos/efectos adversos , Canales de Calcio/metabolismo , Calcio/metabolismo , Mucosa Intestinal/metabolismo , Receptores Sensibles al Calcio/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Calcio/agonistas , Calcio/sangre , Canales de Calcio/genética , Cinacalcet/efectos adversos , Modelos Animales de Enfermedad , Estrenos/farmacología , Femenino , Técnicas de Sustitución del Gen , Humanos , Hipercalcemia/inducido químicamente , Hipercalcemia/prevención & control , Hiperparatiroidismo Secundario/inducido químicamente , Hiperparatiroidismo Secundario/tratamiento farmacológico , Hipocalcemia/inducido químicamente , Hipocalcemia/tratamiento farmacológico , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Túbulos Renales/metabolismo , Masculino , Ratones , Ratones Transgénicos , Oocitos , Hormona Paratiroidea/metabolismo , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/farmacología , Pirrolidinonas/farmacología , Receptores Sensibles al Calcio/agonistas , Canales Catiónicos TRPV/genética , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismo , XenopusRESUMEN
Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca2+ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca2+ , and we show that this Ca2+ -mediated response is contingent on the function of calcium-dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca2+ signalling plays in the invasion biology of apicomplexan parasites.
Asunto(s)
Calcio/metabolismo , Vesículas Citoplasmáticas/metabolismo , Orgánulos/metabolismo , Proteínas Quinasas/metabolismo , Toxoplasma/metabolismo , Calcio/agonistas , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Citoplasma/metabolismo , Exocitosis/genética , Fibroblastos/parasitología , Humanos , Proteínas Quinasas/genética , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/genética , Toxoplasma/patogenicidadRESUMEN
Chlorogenic acid (CGA) is a phenolic compound with various health-promoting properties, including antioxidant effects and a wide range of antibacterial activities. However, the antibacterial mechanism remains unclear. We investigated the underlying mode of action of CGA against Escherichia coli, which shows bacterial apoptosis-like death. Cells treated with CGA showed apoptotic features such as membrane depolarisation, caspase-like protein expression, increased intracellular Ca2+ levels, phosphatidylserine externalisation, and DNA fragmentation. In contrast to common bacterial apoptosis-like death, which is caused by reactive oxygen species (ROS) accumulation, CGA depleted intracellular ROS. Because ROS are important intracellular signalling molecules, and ROS depletion may affect bacterial intracellular signalling pathways, leading to cell death. To determine whether deficiencies in intracellular ROS cause apoptosis-like death, the cells were treated with H2O2 after CGA treatment. H2O2 restored depleted intracellular ROS levels to similar levels as in untreated cells, and cell viability was increased compared to CGA-treated cells. Moreover, apoptotic features were attenuated in H2O2 post-treated cells. These results demonstrate that CGA induces bacterial apoptosis in E. coli and intracellular ROS depletion is a core regulator in the progression of bacterial apoptosis-like death.
Asunto(s)
Antibacterianos/farmacología , Ácido Clorogénico/farmacología , Proteínas de Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Calcio/agonistas , Calcio/metabolismo , Caspasas/genética , Caspasas/metabolismo , Fragmentación del ADN/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expresión Génica , Peróxido de Hidrógeno/farmacología , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Imitación Molecular , Fosfatidilserinas/metabolismo , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/antagonistas & inhibidoresRESUMEN
STUDY QUESTION: Does coumestrol inhibit proliferation of human placental choriocarcinoma cells? SUMMARY ANSWER: Coumestrol promotes cell death in the choriocarcinoma cells by regulating ERK1/2 MAPK and JNK MAPK signaling pathways and through disruption of Ca2+ and ROS homeostasis. WHAT IS KNOWN ALREADY: A number of patients who suffer from choriocarcinomas fail to survive due to delayed diagnosis or a recurrent tumor and resistance to traditional chemotherapy using platinum-based agents and methotrexate. To overcome these limitations, it is important to discover novel compounds which have no adverse effects yet can inhibit the expression of a target molecule to develop, as a novel therapeutic for prevention and/or treatment of choriocarcinomas. STUDY DESIGN, SIZE, DURATION: Effects of coumestrol on human placental choriocarcinoma cell lines, JAR and JEG3, were assessed in diverse assays in a dose- and time-dependent manner. PARTICIPCANTS/MATERIALS, SETTING, METHODS: Effects of coumestrol on cell proliferation, apoptosis (annexin V expression, propidium iodide staining, TUNEL and invasion assays), mitochondria-mediated apoptosis, production of reactive oxygen species (ROS), lipid peroxidation, glutathione levels and endoplasmic reticulum (ER) stress proteins in JAR and JEG3 cells were determined. Signal transduction pathways in JAR and JEG3 cells in response to coumestrol were determined by western blot analyses. MAIN RESULTS AND THE ROLE OF CHANCE: Results of the present study indicated that coumestrol suppressed proliferation and increased apoptosis in JAR and JEG3 cells by inducing pro-apoptotic proteins, Bax and Bak. In addition, coumestrol increased ROS production, as well as lipid peroxidation and glutathione levels in JAR and JEG3 cells. Moreover, coumestrol-induced depolarization of mitochondrial membrane potential (MMP) and increased cytosolic and mitochondrial Ca2+ levels in JAR and JEG3 cells. Consistent with those results, treatment of JAR and JEG3 cells with a Ca2+ chelator and an inhibitor of IP3 receptor decreased coumestrol-induced depolarization of MMP and increased proliferation in JAR and JEG3 cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: A lack of in vivo animal studies is a major limitation of this research. The effectiveness of coumestrol to induce apoptosis of human placental choriocarcinoma cells requires further investigation. WIDER IMPLICATIONS OF THE FINDINGS: Our results indicate that coumestrol induces apoptotic effects on placental choriocarcinoma cells by regulating cell signaling and mitochondrial-mediated functions, with a potential to impair progression of the cancer. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.).
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Cumestrol/farmacología , Células Epiteliales/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Mitocondrias/efectos de los fármacos , Fitoestrógenos/farmacología , Apoptosis/genética , Calcio/agonistas , Calcio/metabolismo , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Corion/efectos de los fármacos , Corion/metabolismo , Corion/patología , Relación Dosis-Respuesta a Droga , Estrés del Retículo Endoplásmico/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Glutatión/metabolismo , Humanos , Peroxidación de Lípido , MAP Quinasa Quinasa 4/genética , MAP Quinasa Quinasa 4/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Proteína Destructora del Antagonista Homólogo bcl-2/agonistas , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/agonistas , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoAsunto(s)
Angina Pectoris Variable/diagnóstico por imagen , Vasoespasmo Coronario/diagnóstico por imagen , Angina Pectoris Variable/tratamiento farmacológico , Angina Pectoris Variable/fisiopatología , Angiografía , Calcio/agonistas , Vasoespasmo Coronario/fisiopatología , Diagnóstico Diferencial , Humanos , Isosorbida/uso terapéutico , Masculino , Persona de Mediana EdadAsunto(s)
Humanos , Masculino , Persona de Mediana Edad , Vasoespasmo Coronario/diagnóstico por imagen , Angina Pectoris Variable/diagnóstico por imagen , Angiografía , Calcio/agonistas , Vasoespasmo Coronario/fisiopatología , Diagnóstico Diferencial , Isosorbida/uso terapéutico , Angina Pectoris Variable/fisiopatología , Angina Pectoris Variable/tratamiento farmacológicoRESUMEN
OBJECTIVES: Differentiation of embryonic stem (ES) cells may be regulated by mechanical strain. Herein, signaling molecules underlying mechanical stimulation of vasculogenesis and expression of angiogenesis guidance cues were investigated in ES cell-derived embryoid bodies. METHODS AND RESULTS: Treatment of embryoid bodies with 10% static mechanical strain using a Flexercell strain system significantly increased CD31-positive vascular structures and the angiogenesis guidance molecules plexinB1, ephrin B2, neuropilin1 (NRP1), semaphorin 4D (sem4D) and robo4 as well as vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) as evaluated by Western blot and real time RT-PCR. In contrast ephrin type 4 receptor B (EphB4) expression was down-regulated upon mechanical strain, indicating an arterial-type differentiation. Robo1 protein expression was modestly increased with no change in mRNA expression. Mechanical strain increased intracellular calcium as well as reactive oxygen species (ROS) and nitric oxide (NO). Mechanical strain-induced vasculogenesis was abolished by the NOS inhibitor L-NAME, the NADPH oxidase inhibitor VAS2870, upon chelation of intracellular calcium by BAPTA as well as upon siRNA inactivation of ephrin B2, NRP1 and robo4. BAPTA blunted the strain-induced expression of angiogenic growth factors, the increase in NO and ROS as well as the expression of NRP1, sem4D and plexinB1, whereas ephrin B2, EphB4 as well as robo1 and robo4 expression were not impaired. CONCLUSIONS: Mechanical strain stimulates vasculogenesis of ES cells by the intracellular messengers ROS, NO and calcium as well as by upregulation of angiogenesis guidance molecules and the angiogenic growth factors VEGF, FGF-2 and PDGF-BB.
Asunto(s)
Calcio/metabolismo , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Miocitos Cardíacos/metabolismo , Óxido Nítrico/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Antígenos CD/genética , Antígenos CD/metabolismo , Becaplermina , Benzoxazoles/farmacología , Fenómenos Biomecánicos , Calcio/agonistas , Diferenciación Celular , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Embrión de Mamíferos , Cuerpos Embrioides/citología , Cuerpos Embrioides/efectos de los fármacos , Efrina-B2/antagonistas & inhibidores , Efrina-B2/genética , Efrina-B2/metabolismo , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , NG-Nitroarginina Metil Éster/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/genética , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1/antagonistas & inhibidores , Neuropilina-1/genética , Neuropilina-1/metabolismo , Óxido Nítrico/agonistas , Proteínas Proto-Oncogénicas c-sis/genética , Proteínas Proto-Oncogénicas c-sis/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/agonistas , Receptor EphB4/genética , Receptor EphB4/metabolismo , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores Inmunológicos/antagonistas & inhibidores , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Semaforinas/genéticaRESUMEN
BACKGROUND/AIMS: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. METHODS: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. RESULTS: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. CONCLUSIONS: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Calcio/agonistas , Retículo Endoplásmico/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Estilbenos/farmacología , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7/genética , Caspasa 7/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células HeLa , Células Endoteliales de la Vena Umbilical Humana , Humanos , Transporte Iónico/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Especificidad de Órganos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Resveratrol , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca(2+) concentration. However, glucose evoked a rapid elevation of intracellular Ca(2+) followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Dispositivos Laboratorio en un Chip , Receptores Acoplados a Proteínas G/metabolismo , Análisis de la Célula Individual/métodos , Percepción del Gusto/efectos de los fármacos , Imagen de Lapso de Tiempo/métodos , Calcio/agonistas , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Células Enteroendocrinas/citología , Células Enteroendocrinas/efectos de los fármacos , Células Enteroendocrinas/metabolismo , Glucosa/antagonistas & inhibidores , Glucosa/farmacología , Gymnema sylvestre/química , Ensayos Analíticos de Alto Rendimiento/instrumentación , Humanos , Modelos Biológicos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Compuestos de Amonio Cuaternario/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Análisis de la Célula Individual/instrumentación , Sacarosa/análogos & derivados , Sacarosa/farmacología , Gusto/efectos de los fármacos , Gusto/fisiología , Percepción del Gusto/fisiología , Imagen de Lapso de Tiempo/instrumentaciónRESUMEN
The effect of protriptyline on Ca2+ physiology in human hepatoma is unclear. This study explored the effect of protriptyline on [Ca2+ ]i and cytotoxicity in HepG2 human hepatoma cells. Protriptyline (50-150 µM) evoked [Ca2+ ]i rises. The Ca2+ entry was inhibited by removal of Ca2+ . Protriptyline-induced Ca2+ entry was confirmed by Mn2+ -induced quench of fura-2 fluorescence. Except nifedipine, econazole, SKF96365, GF109203X, and phorbol 12-myristate 13 acetate did not inhibit Ca2+ entry. Treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) inhibited 40% of protriptyline-induced response. Treatment with protriptyline abolished BHQ-induced response. Inhibition of phospholipase C (PLC) suppressed protriptyline-evoked response by 70%. At 20-40 µM, protriptyline killed cells which was not reversed by the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in HepG2 cells, protriptyline induced [Ca2+ ]i rises that involved Ca2+ entry through nifedipine-sensitive Ca2+ channels and PLC-dependent Ca2+ release from endoplasmic reticulum. Protriptyline induced Ca2+ -independent cell death.
Asunto(s)
Antidepresivos Tricíclicos/farmacología , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Protriptilina/farmacología , Calcio/agonistas , Cationes Bivalentes , Econazol/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Colorantes Fluorescentes , Fura-2 , Células Hep G2 , Humanos , Hidroquinonas/farmacología , Imidazoles/farmacología , Indoles/farmacología , Transporte Iónico/efectos de los fármacos , Cinética , Maleimidas/farmacología , Manganeso/farmacología , Nifedipino/farmacología , Protriptilina/antagonistas & inhibidores , Espectrometría de Fluorescencia , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
The uterine myometrium (UT-myo) is a therapeutic target for preterm labor, labor induction, and postpartum hemorrhage. Stimulation of intracellular Ca2+-release in UT-myo cells by oxytocin is a final pathway controlling myometrial contractions. The goal of this study was to develop a dual-addition assay for high-throughput screening of small molecular compounds, which could regulate Ca2+-mobilization in UT-myo cells, and hence, myometrial contractions. Primary murine UT-myo cells in 384-well plates were loaded with a Ca2+-sensitive fluorescent probe, and then screened for inducers of Ca2+-mobilization and inhibitors of oxytocin-induced Ca2+-mobilization. The assay exhibited robust screening statistics (Z´ = 0.73), DMSO-tolerance, and was validated for high-throughput screening against 2,727 small molecules from the Spectrum, NIH Clinical I and II collections of well-annotated compounds. The screen revealed a hit-rate of 1.80% for agonist and 1.39% for antagonist compounds. Concentration-dependent responses of hit-compounds demonstrated an EC50 less than 10µM for 21 hit-antagonist compounds, compared to only 7 hit-agonist compounds. Subsequent studies focused on hit-antagonist compounds. Based on the percent inhibition and functional annotation analyses, we selected 4 confirmed hit-antagonist compounds (benzbromarone, dipyridamole, fenoterol hydrobromide and nisoldipine) for further analysis. Using an ex vivo isometric contractility assay, each compound significantly inhibited uterine contractility, at different potencies (IC50). Overall, these results demonstrate for the first time that high-throughput small-molecules screening of myometrial Ca2+-mobilization is an ideal primary approach for discovering modulators of uterine contractility.
Asunto(s)
Calcio/metabolismo , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Miometrio/metabolismo , Contracción Uterina , Útero/metabolismo , Útero/fisiología , Animales , Calcio/agonistas , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ratones , Miometrio/citología , Miometrio/efectos de los fármacos , Oxitocina/farmacología , Embarazo , Cultivo Primario de Células , Reproducibilidad de los Resultados , Útero/efectos de los fármacosRESUMEN
Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca2+ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca2+-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca2+ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC50 = 2.64 µM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC50 = 1.89 µM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC50 = 0.33 µM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Hipertensión Pulmonar Primaria Familiar/patología , Músculo Liso Vascular/patología , Arteria Pulmonar/patología , Embolia Pulmonar/patología , Receptores Sensibles al Calcio/antagonistas & inhibidores , Tromboembolia/patología , Compuestos de Anilina/farmacología , Benzamidas/farmacología , Calcio/agonistas , Estudios de Casos y Controles , Células Cultivadas , Enfermedad Crónica , Ciclohexilaminas/farmacología , Hipertensión Pulmonar Primaria Familiar/tratamiento farmacológico , Hipertensión Pulmonar Primaria Familiar/metabolismo , Humanos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Naftalenos/farmacología , Fenetilaminas , Propilaminas , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Embolia Pulmonar/tratamiento farmacológico , Embolia Pulmonar/metabolismo , Tromboembolia/tratamiento farmacológico , Tromboembolia/metabolismoRESUMEN
Ghrelin, a gut and brain peptide, has recently been shown to be involved in motivated behavior and regulation of the sleep and wakefulness cycle. The laterodorsal tegmental nucleus (LDT) is involved in appetitive behavior and control of the arousal state of an organism, and accordingly, behavioral actions of ghrelin could be mediated by direct cellular actions within this nucleus. Consistent with this interpretation, postsynaptically mediated depolarizing membrane actions of ghrelin on LDT neurons have been reported. Direct actions were ascribed solely to closure of a potassium conductance however this peptide has been shown in other cell types to lead to rises in calcium via release of calcium from intracellular stores. To determine whether ghrelin induced intracellular calcium rises in mouse LDT neurons, we conducted calcium imaging studies in LDT brain slices loaded with the calcium binding dye, Fura-2AM. Ghrelin elicited TTX-insensitive changes in dF/F indicative of rises in calcium, and a portion of these rises were independent of membrane depolarization, as they persisted in conditions of high extracellular potassium solutions and were found to involve SERCA-pump mediated intracellular calcium stores. Involvement of the ghrelin receptor (GHR-S) in these actions was confirmed. Taken together with other studies, our data suggest that ghrelin has multiple cellular actions on LDT cells. Ghrelin's induction of calcium via intracellular release in the LDT could play a role in behavioral actions of this peptide as the LDT governs processes involved in stimulation of motivated behavior and control of cortical arousal.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Calcio/metabolismo , Ghrelina/farmacología , Neuronas/efectos de los fármacos , Tegmento Mesencefálico/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Animales Recién Nacidos , Apetito/fisiología , Nivel de Alerta/fisiología , Calcio/agonistas , Colorantes Fluorescentes , Fura-2/análogos & derivados , Ratones , Microtomía , Imagen Molecular , Neuronas/citología , Neuronas/metabolismo , Técnicas de Placa-Clamp , Potasio/metabolismo , Potasio/farmacología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Tegmento Mesencefálico/citología , Tegmento Mesencefálico/metabolismo , Tetrodotoxina/farmacología , Técnicas de Cultivo de TejidosRESUMEN
The excess vascular endothelial growth factor (VEGF) produced in the Alzheimer's disease (AD) brain can harm neurons, blood vessels, and other components of the neurovascular units (NVUs). But could astrocytes partaking in networks of astrocyte-neuron teams and connected to blood vessels of NVUs contribute to VEGF production? We have shown with cultured cerebral cortical normal (i.e., untransformed) adult human astrocytes (NAHAs) that exogenous amyloid-ß peptides (Aßs) stimulate the astrocytes to make and secrete large amounts of Aßs and nitric oxide by a mechanism mediated through the calcium-sensing receptor (CaSR). Here, we report that exogenous Aßs stimulate the NAHAs to produce and secrete even VEGF-A through a CaSR-mediated mechanism. This is indicated by the ability of Aßs to specifically bind the CaSR, and the capability of a CaSR activator, the "calcimimetic" NPS R-568, to imitate, and of the CaSR antagonist, "calcilytic" NPS 2143, to inhibit, the Aßs stimulation of VEGF-A production and secretion by the NAHAs. Thus, Aßs that accumulate in the AD brain may make the astrocytes that envelop and functionally collaborate with neurons into multi-agent AD-driving "machines" via a CaSR signaling mechanism(s). These observations suggest the possibility that CaSR allosteric antagonists such as NPS 2143 might impede AD progression.
Asunto(s)
Péptidos beta-Amiloides/farmacología , Astrocitos/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Receptores Sensibles al Calcio/fisiología , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Adulto , Regulación Alostérica , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Compuestos de Anilina/farmacología , Astrocitos/metabolismo , Calcio/agonistas , Comunicación Celular , Células Cultivadas , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Naftalenos/farmacología , Neuronas/metabolismo , Óxido Nítrico/metabolismo , Fenetilaminas , Propilaminas , Unión Proteica , Receptores Sensibles al Calcio/antagonistas & inhibidores , Lóbulo Temporal/citología , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
To clarify the mechanism(s) underlying intracellular Ca(2+) concentration ([Ca(2+)]i) oscillations induced by an elevation in extracellular Ca(2+) concentration ([Ca(2+)]e) via the extracellular Ca(2+)-sensing receptor (CaR), we analyzed the pattern of [Ca(2+)]i response in multiple (2,303) individual HEK-293 cells transfected with the human CaR. An increase in the [Ca(2+)]e from 1.5 to 3 mM produced oscillatory fluctuations in [Ca(2+)]i in 70% of the cell population. To determine the role of PKC in the generation of [Ca(2+)]i oscillations, cells were exposed to increasing concentrations (0.5-5 µM) of the preferential PKC inhibitor Ro-31-8220 before stimulation by extracellular Ca(2+). Ro-31-8220 at 3-5 µM completely eliminated the [Ca(2+)]e-evoked [Ca(2+)]i oscillations and transformed the pattern to a peak and sustained plateau response. Treatment with other broad PKC inhibitors, including GFI or Gö6983, produced an identical response. Similarly, treatment with Ro-31-8220 or GFI eliminated [Ca(2+)]e-evoked [Ca(2+)]i oscillations in colon-derived SW-480 cells expressing the CaR. Treatment with inhibitors targeting classic PKCs, including Gö6976 and Ro-32-0432 as well as small interfering RNA-mediated knockdown of PKCα, strikingly reduced the proportion of cell displaying [Ca(2+)]e-evoked [Ca(2+)]i oscillations. Furthermore, none of the cells analyzed expressing a CaR mutant in which the major PKC phosphorylation site Thr(888) was converted to alanine (CaRT888A) showed [Ca(2+)]i oscillations after CaR activation. Our results show that [Ca(2+)]i oscillations induced by activation of the CaR in response to an increase in extracellular Ca(2+) or exposure to the calcimimetic R-568 result from negative feedback involving PKCα-mediated phosphorylation of the CaR at Thr(888).
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Proteína Quinasa C-alfa/metabolismo , Receptores Sensibles al Calcio/metabolismo , Compuestos de Anilina/farmacología , Calcio/agonistas , Línea Celular , Inhibidores Enzimáticos/farmacología , Células HEK293 , Humanos , Indoles/farmacología , Transporte Iónico , Maleimidas/farmacología , Fenetilaminas , Fosforilación , Propilaminas , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C-alfa/genética , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
Amniotic fluid-derived stem (AFS) cells have been identified as a promising source for cell therapy applications in bone traumatic and degenerative damage. Calcium Sensing Receptor (CaSR), a G protein-coupled receptor able to bind calcium ions, plays a physiological role in regulating bone metabolism. It is expressed in different kinds of cells, as well as in some stem cells. The bone CaSR could potentially be targeted by allosteric modulators, in particular by agonists such as calcimimetic R-568, which may potentially be helpful for the treatment of bone disease. The aim of our study was first to investigate the presence of CaSR in ovine Amniotic Fluid Mesenchymal Stem Cells (oAFMSCs) and then the potential role of calcimimetics in in vitro osteogenesis. oAFMSCs were isolated, characterized and analyzed to examine the possible presence of CaSR by western blotting and flow cytometry analysis. Once we had demonstrated CaSR expression, we worked out that 1 µM R-568 was the optimal and effective concentration by cell viability test (MTT), cell number, Alkaline Phosphatase (ALP) and Alizarin Red S (ARS) assays. Interestingly, we observed that basal diffuse CaSR expression in oAFMSCs increased at the membrane when cells were treated with R-568 (1 µM), potentially resulting in activation of the receptor. This was associated with significantly increased cell mineralization (ALP and ARS staining) and augmented intracellular calcium and Inositol trisphosphate (IP3) levels, thus demonstrating a potential role for calcimimetics during osteogenic differentiation. Calhex-231, a CaSR allosteric inhibitor, totally reversed R-568 induced mineralization. Taken together, our results demonstrate for the first time that CaSR is expressed in oAFMSCs and that calcimimetic R-568, possibly through CaSR activation, can significantly improve the osteogenic process. Hence, our study may provide useful information on the mechanisms regulating osteogenesis in oAFMSCs, perhaps prompting the use of calcimimetics in bone regenerative medicine.
Asunto(s)
Líquido Amniótico/citología , Compuestos de Anilina/farmacología , Diferenciación Celular/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Receptores Sensibles al Calcio/metabolismo , Fosfatasa Alcalina/metabolismo , Compuestos de Anilina/química , Animales , Benzamidas/farmacología , Western Blotting , Calcio/agonistas , Calcio/metabolismo , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ciclohexilaminas/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Inositol 1,4,5-Trifosfato/metabolismo , Células Madre Mesenquimatosas/citología , Osteogénesis/efectos de los fármacos , Fenetilaminas , Propilaminas , Receptores Sensibles al Calcio/antagonistas & inhibidores , Ovinos , EstereoisomerismoRESUMEN
Although calcimimetics were developed to block parathyroid hormone synthesis, some reports suggest that they may also reduce blood pressure by unknown mechanisms. Calcimimetic-induced changes in the synthesis of endothelial vasoactive factors could be involved. Wistar rats were treated with the calcimimetic R-568, and systolic blood pressure (SBP) was registered with a tail-cuff sphygmomanometer, the content of endothelial nitric oxide synthase (eNOS) and endothelin-converting enzyme (ECE-1) in tissue was evaluated by immunohistochemistry and Western blot, circulating levels of endothelin-1 (ET-1) were measured by ELISA. R-568 reduced SBP and circulating levels of ET-1, without changes in eNOS expression. In contrast, R-568 increased the lung and vascular content of ECE-1. In order to analyze the mechanisms involved, we studied the effect of R-568 on human endothelial cells. R-568 did not modify neither eNOS protein content nor pre-pro-ET-1 mRNA expression, but increased ECE-1 protein content, and decreased ET-1 synthesis and ECE-1 activity. The inhibition of ECE-1 activity was very strong, similar to the classic ECE inhibitor phosphoramidon, the addition of exogenous zinc restored enzymatic activity. Moreover, the amount of zinc in immunoprecipitated ECE from R-568 treated cells was 3-fold less than in control cells. In conclusion, R-568 inhibits ECE by expelling zinc from the enzyme, with the subsequent decrease in enzymatic activity and reducing circulating levels of ET-1, which may be responsible for the lower SBP observed in R-568-treated rats. This descent would be partially compensated by the increased synthesis of the ECE-1 itself, and by other homeostatic mechanisms that regulate SBP.