RESUMEN
Chromaffin granules isolated from adrenal glands constitute a powerful experimental tool to the study of secretory vesicle components and their participation in fusion and docking processes, vesicle aggregation, and interactions with cytosolic components. Although it is possible to isolate and purify chromaffin granules from adrenal glands of different species, bovine adrenal glands are the most used tissue source due to its easy handling and the large amount of granules that can be obtained from this tissue. In this chapter, we describe an easy-to-use and short-term protocol for efficiently obtaining highly purified chromaffin granules from bovine adrenal medulla. We additionally include protocols to isolate granules from cultured bovine chromaffin cells and PC12 cells, as well as a section to obtain chromaffin granules from mouse adrenal glands.
Asunto(s)
Médula Suprarrenal , Células Cromafines , Células Neuroendocrinas , Glándulas Suprarrenales , Animales , Bovinos , Gránulos Cromafines , Ratones , Células PC12 , RatasRESUMEN
The extent and type of hormones and active peptides secreted by the chromaffin cells of the adrenal medulla have to be adjusted to physiological requirements. The chromaffin cell secretory activity is controlled by the splanchnic nerve firing frequency, which goes from approximately 0.5 Hz in basal conditions to more than 15 Hz in stress. Thus, these neuroendocrine cells maintain a tonic release of catecholamines under resting conditions, massively discharge intravesicular transmitters in response to stress, or adequately respond to moderate stimuli. In order to adjust the secretory response to the stimulus, the adrenal chromaffin cells have an appropriate organization of Ca2+ channels, secretory granules pools, and sets of proteins dedicated to selectively control different steps of the secretion process, such as the traffic, docking, priming and fusion of the chromaffin granules. Among the molecules implicated in such events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, Ca2+ sensors like Munc13 and synaptotagmin-1, chaperon proteins such as Munc18, and the actomyosin complex. In the present review, we discuss how these different actors contribute to the extent and maintenance of the stimulus-dependent exocytosis in the adrenal chromaffin cells.
Asunto(s)
Médula Suprarrenal/metabolismo , Gránulos Cromafines/metabolismo , Exocitosis , Animales , Canales de Calcio/metabolismo , Humanos , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Animals living in nontropical climates modify their physiology and behavior to adapt to seasonal environmental changes. Part of this adaptation involves the release of catecholamine from sympathetic nerve endings and the adrenal medulla, which play a major role in regulating energy balance. The aim of this work was to investigate whether adult male viscachas in their natural habitat exhibits structural changes in the adrenal medulla during the annual seasonal cycle. In August-September, chromaffin granules revealed ultrastructural changes suggestive of piecemeal degranulation. Quantitative morphometric analysis by transmission electron microscopy showed a significantly lower percentage of resting chromaffin granules and a higher percentage of altered granules and empty containers in August-September (late winter) compared to February-March (late summer), suggesting an increased secretory process of catecholamines in August-September. The mechanism of piecemeal degranulation might amplify this process, encouraging the adaptive response to winter environmental conditions. Tissue levels of epinephrine, norepinephrine, and dopamine (analyzed by high-performance liquid chromatography) changed throughout the year, reaching maximum values in February-March and minimum values in August-September. These results demonstrate morphological and biochemical seasonal variations of the adrenal medulla, suggesting that epinephrine might promote energy mobilization, which allow the Lagostomus to cope with adverse environmental conditions and thus to survive during winter season.
Asunto(s)
Médula Suprarrenal/metabolismo , Catecolaminas/metabolismo , Gránulos Cromafines/metabolismo , Roedores/metabolismo , Estaciones del Año , Adaptación Fisiológica , Médula Suprarrenal/ultraestructura , Animales , Degranulación de la Célula , Gránulos Cromafines/ultraestructura , Cromatografía Líquida de Alta Presión , Dopamina/metabolismo , Metabolismo Energético , Epinefrina/metabolismo , Masculino , Microscopía Electrónica de Transmisión , Norepinefrina/metabolismo , Lluvia , Luz Solar , Temperatura , Factores de TiempoRESUMEN
Although synaptophysin is one of the most abundant integral proteins of synaptic vesicle membranes, its contribution to neurotransmitter release remains unclear. One possibility is that through its association with dynamin it controls the fine tuning of transmitter release. To test this hypothesis, we took advantage of amperometric measurements of quantal catecholamine release from chromaffin cells. First, we showed that synaptophysin and dynamin interact in chromaffin granule-rich fractions and that this interaction relies on the C terminal of synaptophysin. Experimental maneuvers that are predicted to disrupt the association between these two proteins, such as injection of antibodies against dynamin or synaptophysin, or peptides homologous to the C terminal of synaptophysin, increased the quantal size and duration of amperometric spikes. In contrast, the amperometric current that precedes the spike remained unchanged, indicating that synaptophysin/dynamin association does not regulate the initial fusion pore, but it appears to target a later step of exocytosis to control the amount of catecholamines released during a single vesicle fusion event.
Asunto(s)
Células Cromafines/metabolismo , Dinaminas/metabolismo , Exocitosis/fisiología , Sinaptofisina/metabolismo , Animales , Anticuerpos/farmacología , Bovinos , Células Cultivadas , Células Cromafines/ultraestructura , Gránulos Cromafines/efectos de los fármacos , Gránulos Cromafines/metabolismo , Dinaminas/genética , Dinaminas/inmunología , Electroquímica/métodos , Exocitosis/efectos de los fármacos , Inmunoprecipitación/métodos , Microinyecciones , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Sinaptofisina/química , Sinaptofisina/genética , Sinaptofisina/inmunología , Proteína 2 de Membrana Asociada a Vesículas/metabolismoRESUMEN
This work presents the structure and ultrastructure of the interrenal gland and chromaffin cells, as well as the morphology of the head kidney of Brycon cephalus. The head kidney is composed of fused bilateral lobes located anterior to the swim bladder and ventrolateral to the spinal column. The parenchyma revealed lympho-haematopoietic tissue, melano-macrophage centres, interrenal gland and chromaffin cells. The interrenal gland consisted of cords or strands of cells grouped around the posterior cardinal vein and their branches. Chromaffin cells are found in small groups, closely associated with the interrenal gland and/or under the endothelium of the posterior cardinal vein. So far, the ultrastructural analysis has revealed only one interrenal cell type which contained abundant smooth endoplasmic reticulum and numerous mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Two types of chromaffin cells were observed. The first type was characterized by the presence of vesicles with round, strongly electron-dense granules, which were eccentrically located. Such cells were interpreted as noradrenaline cells. Meanwhile, cells which contained smaller vesicles and electron-lucent granules, with a small halo separating the granule from the vesicular limiting membrane, were identified as adrenaline cells.
Asunto(s)
Células Cromafines/ultraestructura , Peces/anatomía & histología , Glándula Interrenal/citología , Animales , Gránulos Cromafines/ultraestructura , Sistema Cromafín/ultraestructura , Glándula Interrenal/ultraestructura , Microscopía ElectrónicaRESUMEN
The effect of oxytocin (0.25 IU/100 g per day) on the adrenal medulla was examined in intact, intact estrogen-treated, castrated and castrated testosterone-treated adult male Wistar rats. Stereological analysis of the gland (N = 5 rats per group) revealed that in intact animals the number of chromaffin cells (x10(3)) was significantly increased after 3-day (saline: 467.6 +/- 27.4; oxytocin: 567.6 +/- 28.9) or 7-day (saline: 486.2 +/- 39.1; oxytocin: 618.7 +/- 36.8) oxytocin administration. During 7 days of recovery after the 7-day treatment, the chromaffin cell number returned to the control level (saline: 491.4 +/- 12.6; oxytocin: 554.4 +/- 28.7). The effect of oxytocin on chromaffin cell number was also observed in rats simultaneously injected with estradiol (0.3 micrograms/100 g per day) for 10 days (estradiol: 454.3 +/- 32.8; estradiol+oxytocin: 576.1 +/- 25.0), as well as in 10-day castrated rats (saline: 594.7 +/- 22.7; oxytocin: 765.3 +/- 33.1). Testosterone replacement (0.6 mg/100 g per day) abolished the medullary response to oxytocin (testosterone+saline: 528.5 +/- 24.7; testosterone+oxytocin: 620.8 +/- 56.0). There was a 20% rise in adrenal dopamine content (from 0.236 +/- 0.015 to 0.283 +/- 0.015 microgram per pair of glands; N = 9-12) in intact rats injected with oxytocin for 3 days. Oxytocin had no effect on any of the catecholamine levels in adrenal glands of rats exposed to stress induced by constant lighting. The present data indicate that the proliferative response of chromaffin tissue to oxytocin depends on the gonadal hormone level and the basal activity of the adrenal medulla.
Asunto(s)
Médula Suprarrenal/efectos de los fármacos , Oxitocina/farmacología , Testosterona/administración & dosificación , Médula Suprarrenal/anatomía & histología , Médula Suprarrenal/fisiología , Animales , Gránulos Cromafines/efectos de los fármacos , Iluminación , Masculino , Oxitocina/administración & dosificación , Ratas , Ratas Wistar , Estrés Fisiológico/complicaciones , Estrés Fisiológico/fisiopatología , Testosterona/sangreRESUMEN
The effect of oxytocin (0.25 IU/100 g per day) on the adrenal medulla was examined in intact, intact estrogen-treated, castrated and castrated testosterone-treated adult male Wistar rats. Stereological analysis of the gland (N = 5 rats per group) revealed that in intact animals the number of chromaffin cells (x10(3)) was significantly increased after 3-day (saline: 467.6 +/- 27.4; oxytocin: 567.6 +/- 28.9) or 7-day (saline: 486.2 +/- 39.1; oxytocin: 618.7 +/- 36.8) oxytocin administration. During 7 days of recovery after the 7-day treatment, the chromaffin cell number returned to the control level (saline: 491.4 +/- 12.6; oxytocin: 554.4 +/- 28.7). The effect of oxytocin on chromaffin cell number was also observed in rats simultaneously injected with estradiol (0.3 microg/100 g per day) for 10 days (estradiol: 454.3 +/- 32.8; estradiol + oxytocin: 576.1 +/- 25.0), as well as in 10-day castrated rats (saline: 594.7 +/- 22.7; oxytocin: 765.3 +/- 33.1). Testosterone replacement (0.6 mg/100 g per day) abolished the medullary response to oxytocin (testosterone + saline: 528.5 +/- 24.7; testosterone + oxytocin: 620.8 +/- 56.0). There was a 20 percent rise in adrenal dopamine content (from 0.236 +/- 0.015 to 0.283 +/- 0.015 microg per pair of glands; N = 9-12) in intact rats injected with oxytocin for 3 days. Oxytocin had no effect on any of the catecholamine levels in adrenal glands of rats exposed to stress induced by constant lighting. The present data indicate that the proliferative response of chromaffin tissue to oxytocin depends on the gonadal hormone level and the basal activity of the adrenal medulla.
Asunto(s)
Masculino , Animales , Ratas , Médula Suprarrenal/efectos de los fármacos , Oxitocina/farmacología , Testosterona/administración & dosificación , Médula Suprarrenal/anatomía & histología , Médula Suprarrenal/fisiología , Gránulos Cromafines/efectos de los fármacos , Iluminación , Oxitocina/administración & dosificación , Ratas Wistar , Estrés Fisiológico/complicaciones , Estrés Fisiológico/fisiopatología , Testosterona/sangreRESUMEN
Exposure of "in vitro" grown immature and adult adrenal chromaffin cells to concentrations of 10(-3) or 10(-5) M but not 10(-7) M GM1 ganglioside, resulted in significant increase in cell diameter, coupled with reduction of adhesion to substrate within 48 hrs of exposure. None of the GM1 concentrations, with or without serum supplementation, did significantly increase neuritogenesis in chromaffin cells. Immature chromaffin cells underwent neuritogenesis when grown in co-cultures with actively growing astroglia from striatum or cerebral cortex, an effect that was potentiated by NGF administration and blocked by anti-NGF. In neither of the former conditions did 10(-6) M GM1 prove to increase the number of neurite emitting cells nor their mean neuritic length further. It is speculated that GM1 does not perform the neuritogenic role described for central neurons in chromaffin cells, nor does it potentiate NGF effect on neuritogenesis observed in other peripheral neurons.
Asunto(s)
Gránulos Cromafines/efectos de los fármacos , Gangliósido G(M1)/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Factores de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Desde noviembre de 1978 a junio de 1991 tratamos a 10 pacientes que presentaban un carcinoide de localización apendícular. Pertenecían al sexo masculino 6 pacientes y 4 al femenino, con una edad promedio de 42 años (rango de 20-68). En 9 pacientes se realizó una apendicectomía y en 1 una hemicolectomía derecha por otra patología colónica asociada. En ningún caso se realizó un diagnóstico preoperatorio y en 3 se sospechó patología apendícular durante otra intervención quirúrgica. En 7 casos el diagnóstico preoperatorio fue apendicitis aguda, sin embargo, el estudio anátomo-patológico no reveló inflamación apendícular en 5 casos. Ningún paciente presentó un síndrome carcinoide. Todos los tumores fueron menores 1 cm., en 9 casos se ubicaban en la punta y 1 en la base (a este último se le efectuó una hemicolectomía derecha por presentar una patología asociada). Pertenecían al tipo clásico 8 pacientes, y 2 al tipo tubular. No se registró ningún caso con células globosas, formas mixtas o asociadas con adenocarcinomas. El seguimiento se realizó durante una media de 7 años (rango de 1-14) sin registrar mortalidad. Consideramos importante para adecuar la terapéutica y el seguimiento tener en cuenta el tamaño la ubicación y la variedad histológica
Asunto(s)
Humanos , Masculino , Femenino , Adolescente , Adulto , Persona de Mediana Edad , Anciano , Tumor Carcinoide , Gránulos Cromafines/patología , Apendicitis/cirugía , Apéndice/patología , Apendicectomía , Adenocarcinoma , Neoplasias del ColonRESUMEN
Desde noviembre de 1978 a junio de 1991 tratamos a 10 pacientes que presentaban un carcinoide de localización apendícular. Pertenecían al sexo masculino 6 pacientes y 4 al femenino, con una edad promedio de 42 años (rango de 20-68). En 9 pacientes se realizó una apendicectomía y en 1 una hemicolectomía derecha por otra patología colónica asociada. En ningún caso se realizó un diagnóstico preoperatorio y en 3 se sospechó patología apendícular durante otra intervención quirúrgica. En 7 casos el diagnóstico preoperatorio fue apendicitis aguda, sin embargo, el estudio anátomo-patológico no reveló inflamación apendícular en 5 casos. Ningún paciente presentó un síndrome carcinoide. Todos los tumores fueron menores 1 cm., en 9 casos se ubicaban en la punta y 1 en la base (a este último se le efectuó una hemicolectomía derecha por presentar una patología asociada). Pertenecían al tipo clásico 8 pacientes, y 2 al tipo tubular. No se registró ningún caso con células globosas, formas mixtas o asociadas con adenocarcinomas. El seguimiento se realizó durante una media de 7 años (rango de 1-14) sin registrar mortalidad. Consideramos importante para adecuar la terapéutica y el seguimiento tener en cuenta el tamaño la ubicación y la variedad histológica
Asunto(s)
Humanos , Masculino , Femenino , Adolescente , Adulto , Persona de Mediana Edad , Apéndice/patología , Apendicitis/cirugía , Tumor Carcinoide , Gránulos Cromafines/patología , Adenocarcinoma , Apendicectomía , Neoplasias del ColonRESUMEN
The association of endogenous synenkephalin and met-enkephalin containing peptides with the membrane of bovine chromaffin granules and physicochemical characteristics of this association were studied. The associated materials were only released at a non physiological pH range and this effect was enhanced with growing salt concentrations (0.5, 1.0 and 2.0 M KSCN). A higher peptide dissociation occurred with membrane solubilizing agents (SDS greater than Triton X-100 greater than digitonin). In microsomes the materials dissociated with 2 M KSCN (pH 7.4) corresponded to peptides larger than 12.0 kDa, while in granules corresponded to molecules smaller than 8.5 kDa, displaying synenkephalin and met-enkephalin immunoreactivities. These data suggest that some sequence of the C-terminal portion of synenkephalin may be responsible for the association of proenkephalin derived peptides with microsome and granule membranes.