RESUMEN
Enterochromaffin cells are the major site of serotonin (5-HT) synthesis and secretion providing â¼95% of the body's total 5-HT. 5-HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5-HT per exocytosis event compared to other endocrine cells. We utilized a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5-HT packaging and release. Using the fluorescent probe FFN511, we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5-HT loading into vesicles. Carbon fiber amperometry studies illustrate that the amount of 5-HT released per exocytosis event from EC cells is dependent on both VMAT and the H(+)-ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5-HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different-sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5-HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5-HT release capacity from individual vesicles.
Asunto(s)
Células Enterocromafines/metabolismo , Vesículas Secretoras/metabolismo , Serotonina/biosíntesis , Corteza Suprarrenal/citología , Corteza Suprarrenal/metabolismo , Animales , Exocitosis/genética , Cobayas , Fusión de Membrana/genética , Ratones , Serotonina/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/genética , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
Mitochondria are the primary site of cellular energy generation and reactive oxygen species (ROS) accumulation. Elevated ROS levels are detrimental to normal cell function and have been linked to the pathogenesis of neurodegenerative disorders such as Down's syndrome (DS) and Alzheimer's disease (AD). RCAN1 is abundantly expressed in the brain and overexpressed in brain of DS and AD patients. Data from nonmammalian species indicates that increased RCAN1 expression results in altered mitochondrial function and that RCAN1 may itself regulate neuronal ROS production. In this study, we have utilized mice overexpressing RCAN1 (RCAN1(ox)) and demonstrate an increased susceptibility of neurons from these mice to oxidative stress. Mitochondria from these mice are more numerous and smaller, indicative of mitochondrial dysfunction, and mitochondrial membrane potential is altered under conditions of oxidative stress. We also generated a PC12 cell line overexpressing RCAN1 (PC12(RCAN1)). Similar to RCAN1(ox) neurons, PC12(RCAN1) cells have an increased susceptibility to oxidative stress and produce more mitochondrial ROS. This study demonstrates that increasing RCAN1 expression alters mitochondrial function and increases the susceptibility of neurons to oxidative stress in mammalian cells. These findings further contribute to our understanding of RCAN1 and its potential role in the pathogenesis of neurodegenerative disorders such as AD and DS.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitocondrias/metabolismo , Proteínas Musculares/metabolismo , Estrés Oxidativo , Animales , Supervivencia Celular/efectos de los fármacos , Proteínas de Unión al ADN , Femenino , Peróxido de Hidrógeno/toxicidad , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Modelos Biológicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The major source of serotonin (5-HT) in the body is the enterochromaffin (EC) cells lining the intestinal mucosa of the gastrointestinal tract. Despite the fact that EC cells synthesise â¼95% of total body 5-HT, and that this 5-HT has important paracrine and endocrine roles, no studies have investigated the mechanisms of 5-HT release from single primary EC cells. We have developed a rapid primary culture of guinea-pig and human EC cells, allowing analysis of single EC cell function using electrophysiology, electrochemistry, Ca(2+) imaging, immunocytochemistry and 3D modelling. Ca(2+) enters EC cells upon stimulation and triggers quantal 5-HT release via L-type Ca(2+) channels. Real time amperometric techniques reveal that EC cells release 5-HT at rest and this release increases upon stimulation. Surprisingly for an endocrine cell storing 5-HT in large dense core vesicles (LDCVs), EC cells release 70 times less 5-HT per fusion event than catecholamine released from similarly sized LDCVs in endocrine chromaffin cells, and the vesicle release kinetics instead resembles that observed in mammalian synapses. Furthermore, we measured EC cell density along the gastrointestinal tract to create three-dimensional (3D) simulations of 5-HT diffusion using the minimal number of variables required to understand the physiological relevance of single cell 5-HT release in the whole-tissue milieu. These models indicate that local 5-HT levels are likely to be maintained around the activation threshold for mucosal 5-HT receptors and that this is dependent upon stimulation and location within the gastrointestinal tract. This is the first study demonstrating single cell 5-HT release in primary EC cells. The mode of 5-HT release may represent a unique mode of exocytosis amongst endocrine cells and is functionally relevant to gastrointestinal sensory and motor function.
Asunto(s)
Calcio/fisiología , Células Enterocromafines/fisiología , Serotonina/fisiología , Animales , Canales de Calcio Tipo L/fisiología , Células Cultivadas , Tracto Gastrointestinal/citología , Cobayas , Humanos , Cinética , Modelos BiológicosRESUMEN
Copper (Cu) is an essential biometal involved in a number of cell functions. Abnormal Cu homeostasis has been identified as a major factor in a number of neurodegenerative disorders. However, little is known about how cells of brain origin maintain Cu homeostasis and in particular, how they respond to an elevated Cu environment. Understanding these processes is essential to obtaining a greater insight into the pathological changes in neurodegeneration and ageing. Although previous studies have shown that Cu in neurons can be associated with synaptic function, there is little understanding of how Cu modulates the regulated secretory vesicle pathways in these cells. In this study, we examined the effect of elevated intracellular Cu on proteins associated with the regulated secretory vesicle pathway in NGF-differentiated PC12 cells that exhibit neuronal-like properties. Increasing intracellular Cu with a cell-permeable Cu-complex (Cu(II)(gtsm)) resulted in increased expression of synaptophysin and robust translocation of this and additional vesicular proteins from synaptic-like microvesicle (SLMV) fractions to chromogranin-containing putative large dense core vesicle (LDCV) fractions in density gradient preparations. The LDCV fractions also contained substantially elevated Cu levels upon treatment of cells with Cu(II)(gtsm). Expression of the H(+) pump, V-ATPase, which is essential for vesicle maturation, was increased in Cu-treated cells while inhibition of V-ATPase prevented translocation of synaptophysin to LDCV fractions. Cu treatment was found to inhibit release of LDCVs in chromaffin cells due to reduced Ca(2+)-mediated vesicle exocytosis. Our findings demonstrate that elevated Cu can modulate LDCV metabolism potentially resulting in sequestration of Cu in this vesicle pool.
Asunto(s)
Cobre/farmacología , Vías Secretoras/efectos de los fármacos , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Cobre/metabolismo , Células PC12 , RatasRESUMEN
RCAN1 is a chromosome 21 gene that controls secretion in endocrine cells, regulates mitochondrial function, and is sensitive to oxidative stress. Regulator of calcineurin 1 (RCAN1) is also an endogenous inhibitor of the protein phosphatase calcineurin, the inhibition of which leads to hypoinsulinemia and diabetes in humans and mice. However, the presence or the role of RCAN1 in insulin-secreting ß-cells and its potential role in the pathogenesis of diabetes is unknown. Hence, the aim of this study is to investigate the presence of RCAN1 in ß-cells and identify its role in ß-cell function. RCAN1 is expressed in mouse islets and in the cytosol of pancreatic ß-cells. We find RCAN1 is a glucose-responsive gene with a 1.5-fold increase in expression observed in pancreatic islets in response to chronic hyperglycemia. The overexpression of the human RCAN1.1 isoform in mice under the regulation of its endogenous promoter causes diabetes, age-associated hyperglycemia, reduced glucose tolerance, hypoinsulinemia, loss of ß-cells, reduced ß-cell insulin secretion, aberrant mitochondrial reactive oxygen species production, and the down-regulation of key ß-cell genes. Our data therefore identifies a novel molecular link between the overexpression of RCAN1 and ß-cell dysfunction. The glucose-responsive nature of RCAN1 provides a potential mechanism of action associated with the ß-cell dysfunction observed in diabetes.
Asunto(s)
Diabetes Mellitus/metabolismo , Intolerancia a la Glucosa/metabolismo , Hiperglucemia/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Musculares/metabolismo , Animales , Proteínas de Unión al Calcio , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/patología , Hiperglucemia/genética , Hiperglucemia/patología , Secreción de Insulina , Células Secretoras de Insulina/patología , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Musculares/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We have previously reported a heteroplasmic mtDNA mutation (T1095C) in the 12SrRNA gene of an Italian family with features of maternally-inherited parkinsonism, antibiotic-mediated deafness and peripheral neuropathy. In the present study, we demonstrate that a transmitochondrial cybrid line derived from the proband of this family shows selective depletion of mitochondrial glutathione and decreases in the activity of complex II/III. Moreover, when exposed to an aminoglycoside antibiotic these cells responded with a ten-fold increase in the number of apoptotic cells compared to controls. These results support a pathogenic role for the T1095C mutation and indicate that the mutation increases the risk for aminoglycoside-induced toxicity.