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BACKGROUND: Tyrosine hydroxylase (TH) catalyzes the rate-limiting step for the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline. Although its distribution in different organs, species and stages of development has been the subject of numerous studies, the recent emergence of 3D imaging techniques has created the potential to shed new light on the dynamics of TH expression during the development of the mammalian central and peripheral nervous systems. NEW METHOD: Here, we describe a flowchart summarizing different protocols adapted to developmental stage-specific tissues to generate a 3D atlas of the catecholaminergic system in the brain and peripheral nervous system in mice from embryonic to pre-weaning stages. The procedures described allowed a quantitative assessment of developing TH-positive neuronal populations and pathways, previously understudied due to dimensional limitations. RESULTS: Our approach allowed us to reveal in 3D the dynamics of the onset and the establishment of the catecholaminergic system in embryonic and developing central and peripheral nervous system. Quantitative analyses applied to 3D images yielded accurate measurements of neuron population volumes and numbers, and tract pathway dimensions for selected TH-positive brain structures. COMPARISON WITH EXISTING METHODS: We applied a set of different protocols to yield a comprehensive flowchart for 3D imaging and a precise quantitative assessment of specific neuronal populations during the course of their development up to adulthood in mice. CONCLUSION: The procedures described and the extensive 3D mapping of TH immunoreactivity at early embryonic and postnatal stages provide a comprehensive view of the onset and development of the catecholaminergic system in the mouse brain and sympathoadrenal nervous system.
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Encéfalo , Tirosina 3-Monooxigenasa , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Catecolaminas , Femenino , Ratones , Embarazo , Diseño de Software , Tirosina 3-Monooxigenasa/metabolismo , DesteteRESUMEN
Granins and their derived-peptides are useful markers of secretion from normal and tumoral neuroendocrine cells. The need to identify new diagnostic markers for neuroendocrine tumors, including pituitary tumors prompted us to determine plasma levels of the secretogranin II-derived peptide EM66 in healthy volunteers with different gonadotroph status and to evaluate its usefulness as a circulating marker for the diagnosis of gonadotroph tumor. Using a radioimmunoassay, we determined plasma EM66 concentrations in healthy men and women volunteers in different physiological conditions in relation with the gonadotroph function. Our results revealed that in men, in women with or without contraception, in pregnant or post-menopausal women, plasma EM66 concentrations are not significantly different, and did not show any correlation with gonadotropin levels. In addition, stimulation or inhibition tests of the gonadotroph axis had no effect on EM66 levels, whatever the group of healthy volunteers investigated while gonadotropin levels showed the expected variations. Immunohistochemical experiments and HPLC analysis showed the occurrence of EM66 in pituitary gonadotroph, lactotroph and corticotroph tumors but not in somatotroph tumor. In patients with gonadotroph or lactotroph tumor, plasma EM66 levels were 1.48 (0.82-4.38) ng/ml and 2.49 (1.19-3.54) ng/ml, respectively. While median value of EM66 was significantly lower in patients with gonadotroph tumor compared to healthy volunteers [2.59 (0.62-4.95) ng/ml], plasma EM66 concentrations were in the same range as normal values and did not show any correlation with gonadotropin levels. These results show that plasma EM66 levels are independent of the activity of the gonadotroph axis in healthy volunteers and, while EM66 levels are reduced in gonadotroph tumors, plasma EM66 does not provide a helpful marker for the diagnosis of these tumors.
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Granins and their derived peptides are valuable circulating biological markers of neuroendocrine tumors. The aim of the present study was to investigate the tumoral chromogranin A (CgA)-derived peptide WE-14 and the potential advantage to combine plasma WE-14 detection with the EM66 assay and the existing current CgA assay for the diagnosis of pheochromocytoma. Compared to healthy volunteers, plasma WE-14 levels were 5.4-fold higher in patients with pheochromocytoma, but returned to normal values after surgical resection of the tumor. Determination of plasma CgA and EM66 concentrations in the same group of patients revealed that the test assays for these markers had an overall 84% diagnostic sensitivity, which is identical to that determined for WE-14. However, we found that WE-14 measurement improved the diagnostic sensitivity when combined with the results of CgA or EM66 assays. By combining the results of the three assays, the sensitivity for the diagnosis of pheochromocytoma was increased to 95%. In fact, the combination of WE-14 with either CgA or EM66 test assays achieved 100% sensitivity for the diagnosis of paragangliomas and sporadic or malignant pheochromocytomas if taken separately to account for the heterogeneity of the tumor. These data indicate that WE-14 is produced in pheochromocytoma and secreted into the general circulation, and that elevated plasma WE-14 levels are correlated with the occurrence of this chromaffin cell tumor. In addition, in association with other biological markers, such as CgA and/or EM66, WE-14 measurement systematically improves the diagnostic sensitivity for pheochromocytoma. These findings support the notion that granin-processing products may represent complementary tools for the diagnosis of neuroendocrine tumors.
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Neoplasias de las Glándulas Suprarrenales/sangre , Cromogranina A/sangre , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/sangre , Tumores Neuroendocrinos/sangre , Péptidos/química , Feocromocitoma/sangre , Adulto , Anciano , Biomarcadores/sangre , Células Cromafines/citología , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Mutación , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenRESUMEN
CONTEXT: A number of incidentally discovered pheochromocytomas are not associated with hypertension. The characteristics of normotensive incidentally discovered pheochromocytomas (NIPs) are poorly known. OBJECTIVE: The purpose of this work was to assess the clinical, hormonal, histological, and molecular features of NIPs. DESIGN: This was a retrospective cohort recruited from 2001 to 2011 in 2 tertiary care medical departments. PATIENTS AND METHODS: Clinical, biological, and radiological investigations performed in 96 consecutive patients with sporadic unilateral pheochromocytomas were examined; 47 patients had overt pheochromocytomas responsible for hypertension. Among the patients with incidental pheochromocytomas, 28 had hypertension and 21 were normotensive (NIPs). A total of 62 tumors were examined to determine the Pheochromocytoma of the Adrenal Gland Scale Score, and 29 were studied for the expression of 16 genes involved in chromaffin cell function. RESULTS: Tumor size and metaiodobenzylguanidine (MIBG) scintigraphy results were similar for hypertensive pheochromocytomas (HPs) and NIPs. Patients with NIPs displayed reduced summed levels of urinary catecholamines and metanephrines and, more specifically, reduced levels of adrenaline and metadrenaline compared with those of patients with HPs (P < .001). Urinary metanephrines had 98% diagnostic sensitivity in patients with HPs and only 75% in patients with NIPs (P < .01). Tumor diameter positively correlated with the total amount of urinary concentrations of metanephrines in patients with HPs (P < .001) but not in patients with NIPs. NIPs displayed global decreased chromaffin gene expression (reaching significance for 5 of them) and 2 corresponding proteins (phenylethanolamine N-methyltransferase and secretogranin II) and a significant increase in the cellularity, mitotic activity, and presence of atypical mitosis (P < .05). CONCLUSIONS: NIPs differ from pheochromocytomas responsible for hypertension and display features of altered chromaffin differentiation. These tumors may be misdiagnosed with the use of the usual biological diagnostic tools.
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Neoplasias de las Glándulas Suprarrenales , Células Cromafines/diagnóstico por imagen , Células Cromafines/fisiología , Regulación Neoplásica de la Expresión Génica , Feocromocitoma , 3-Yodobencilguanidina , Neoplasias de las Glándulas Suprarrenales/diagnóstico por imagen , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/metabolismo , Células Cromafines/patología , Femenino , Humanos , Hipertensión/diagnóstico por imagen , Hipertensión/genética , Hipertensión/metabolismo , Hallazgos Incidentales , Masculino , Persona de Mediana Edad , Feocromocitoma/diagnóstico por imagen , Feocromocitoma/genética , Feocromocitoma/metabolismo , Cintigrafía , Radiofármacos , Estudios Retrospectivos , TranscriptomaRESUMEN
EM66 is a secretogranin II-derived peptide strongly expressed within hypothalamic neuroendocrine areas such as the parvocellular aspect of the paraventricular nucleus (pPVN) as well as the median eminence (ME), suggesting a hypophysiotropic role for this neuropeptide. The aim of the present study was to explore such a role in the corticotrope and thyrotrope axes. We analyzed EM66 occurrence respectively in CRH and TRH neurosecretory cells of the rat pPVN by double immunohistochemistry. Functionally, we studied the effect of acute stress (immobilization for 2 h or cold exposure at 5°C for 4 h) and hypothyroidism (induced by 1-week thyroidectomy) on EM66 immunoreactivity (IR) within the pPVN. Double immunohistochemical labeling revealed that EM66-IR colocalized with CRH or TRH labelings within pPVN hypophysiotropic neurons as well as the axon terminals of the external layer of the ME. Because TRH neuronal population of the pPVN is completely distinct from the CRH neurosecretory system, our data demonstrate the existence of at least two distinct EM66 neuronal populations in the rat pPVN. Acute immobilization or cold exposure stresses did not affect EM66 expression as evaluated by the number of EM66-IR neurons within the pPVN. These results suggest that EM66 does not participate to the phenotypic plasticity of hypothalamic parvocellular neurons in response to acute stress. In addition, short-term hypothyroidism did not provoke any significant variation of the number of intraparaventricular EM66 neurons, indicating that EM66 expression would be insensitive to short-term hypothyroidism despite its occurrence within TRH neurons. Thus, the present data show the occurrence of EM66 in distinct areas of the rat PVN but its expression is not coregulated with those of CRH and TRH during acute stress and hypothyroidism.
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Hormona Liberadora de Corticotropina/metabolismo , Núcleo Hipotalámico Paraventricular/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Secretogranina II/farmacología , Estrés Fisiológico , Tiroidectomía , Hormona Liberadora de Tirotropina/metabolismo , Animales , Inmunohistoquímica , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/citología , Núcleo Hipotalámico Paraventricular/metabolismo , Ratas , Ratas WistarRESUMEN
AIM: Accumulating data suggest that neuropeptides produced by neuroendocrine cells play a crucial role in the progression and aggressiveness of hormone refractory prostate cancer (CaP). In this study, we have investigated the presence and function of the neuropeptide 26RFa in CaP. METHODS: We have localised and quantified tumour cells containing 26RFa and its receptor, GPR103, in CaP sections of various grades. In vitro experiments were performed to investigate the effects of 26RFa on the migration, proliferation and neuroendocrine differentiation of the androgeno-independent (AI) prostate cancer cell line DU145. RESULTS: 26RFa and GPR103 are present in carcinomatous foci exhibiting a neuroendocrine differentiation, and the number of 26RFa and GPR103-immunoreactive cancer cells increases with the grade of CaP. 26RFa stimulated the migration of native or transdifferentiated AI DU145 cells, but had no effect on their proliferation. Furthermore, 26RFa induced the neuroendocrine differentiation of DU145 cells as assessed by the occurrence of neurite-like extensions and the increase of the expression of the neuroendocrine marker chromogranin A. CONCLUSION: The present data indicate that 26RFa may participate to the development of CaP at the AI state by promoting the neuroendocrine differentiation and the migration of cancer cells via autocrine/paracrine mechanisms.
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Neuropéptidos/biosíntesis , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Andrógenos/metabolismo , Diferenciación Celular/fisiología , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Progresión de la Enfermedad , Humanos , Inmunohistoquímica , Masculino , Neoplasias Hormono-Dependientes/metabolismo , Neoplasias Hormono-Dependientes/patología , Células Neuroendocrinas/metabolismo , Células Neuroendocrinas/patología , Receptores Acoplados a Proteínas G/biosíntesisRESUMEN
OBJECTIVES AND METHODS: Neuropeptides, as the main neuroendocrine system effectors, regulate notably the response to different stressors via a secretory plasticity within their respective hypothalamic neuronal populations. The aim of the present study was to explore by immunocytochemistry the occurrence and the potential expression plasticity of the novel neuropeptide EM66 in the CRH neurones of stressed rats. RESULTS: The secretogranin II (SgII)-derived peptide EM66 is strongly expressed within hypothalamic neuroendocrine areas such as the parvocellular aspect of the paraventricular nucleus (pPVN) as well as the median eminence, suggesting a probable hypophysiotropic effect of this peptide. As a first approach to investigate such a role, we evaluated by immunohistochemistry EM66 expression within the pPVN following acute immune stress induced by lipopolysaccharide (LPS) or interleukin-1ß (IL-1ß) injection in rat. This study showed that EM66 is present in the pPVN but the number of EM66 immunolabeled cells did not fluctuate in this structure following LPS peripheral injection. In line with this observation, an intracerebroventricular injection of IL-1ß did not provoke any significant variation of the number of intraparaventricular EM66 neurones. CONCLUSION: The present data revealed for the first time that EM66 expression would be insensitive to the central and peripheral cytokines within the neurosecretory hypothalamic pPVN. This result indicates that EM66 does not participate to the phenotypic plasticity of hypothalamic parvicellular neurones in response to acute inflammatory stress.
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Plasticidad Neuronal , Neuropéptidos/biosíntesis , Núcleo Hipotalámico Paraventricular/metabolismo , Fragmentos de Péptidos/metabolismo , Secretogranina II/metabolismo , Estrés Fisiológico , Animales , Modelos Animales de Enfermedad , Inmunohistoquímica , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Interleucina-1beta , Lipopolisacáridos , Masculino , Núcleo Hipotalámico Paraventricular/inmunología , Fragmentos de Péptidos/inmunología , Ratas , Ratas Wistar , Secretogranina II/química , Secretogranina II/inmunología , Estrés Fisiológico/inmunologíaRESUMEN
Pheochromocytomas are catecholamine-producing tumors arising from chromaffin cells of the adrenal medulla or extra-adrenal location. Along with catecholamines, tumoral cells produce and secrete elevated quantities of trophic peptides which are normally released in a regulated manner by the normal adrenal medulla. Among these peptides, the amounts of pituitary adenylate cyclase-activating polypeptide (PACAP), adrenomedullin (AM), and neuropeptide Y (NPY) are particularly high. These peptides can exert endocrine, paracrine or autocrine effects in numerous cell types. In particular, they have been shown to be involved in cell proliferation and survival, catecholamine production and secretion, and angiogenesis. Some of these processes are exacerbated in pheochromocytomas, raising the possibility of the involvement of trophic peptides. Here, we review the expression levels of NPY, PACAP, and AM and theirs receptors in chromaffin cells and pheochromocytomas, and address their possible implication in the adrenal medulla tumorigenesis and malignant development of pheochromocytomas.
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Neoplasias de las Glándulas Suprarrenales/metabolismo , Células Cromafines/metabolismo , Neuropéptidos/metabolismo , Feocromocitoma/metabolismo , Receptores de Neuropéptido/metabolismo , Neoplasias de las Glándulas Suprarrenales/genética , Animales , Regulación Neoplásica de la Expresión Génica , Humanos , Neuropéptidos/genética , Feocromocitoma/genética , Receptores de Neuropéptido/genéticaRESUMEN
Pheochromocytomas are rare catecholamine-secreting tumors that arise from chromaffin tissue within the adrenal medulla and extra-adrenal sites. Typical clinical manifestations are sustained or paroxysmal hypertension, severe headaches, palpitations and sweating resulting from hormone excess. However, their presentation is highly variable and can mimic many other diseases. The diagnosis of pheochromocytomas depends mainly upon the demonstration of catecholamine excess by 24-h urinary catecholamines and metanephrines or plasma metanephrines. Occurrence of malignant pheochromocytomas can only be asserted by imaging of metastatic lesions, which are associated with a poor survival rate. The characterization of tissue, circulating or genetic markers is therefore crucial for the management of these tumors. Proteins of the granin family and their derived peptides are present in dense-core secretory vesicles and secreted into the bloodstream, making them useful markers for the identification of neuroendocrine cells and neoplasms. In this context, we will focus here on reviewing the distribution and characterization of granins and their processing products in normal and tumoral chromaffin cells, and their clinical usefulness for the diagnosis and prognosis of pheochromocytomas. It appears that, except SgIII, all members of the granin family i.e. CgA, CgB, SgII, SgIV-SgVII and proSAAS, and most of their derived peptides are present in adrenomedullary chromaffin cells and in pheochromocytes. Moreover, besides the routinely used CgA test assays, other assays have been developed to measure concentrations of tissue and/or circulating granins or their derived peptides in order to detect the occurrence of pheochromocytomas. In most cases, elevated levels of these entities were found, in correlation with tumor occurrence, while rarely discriminating between benign and malignant neoplasms. Nevertheless, measurement of the levels of granins and derived peptides improves the diagnostic sensitivity and may therefore provide a complementary tool for the management of pheochromocytomas. However, the existing data need to be substantiated in larger groups of patients, particularly in the case of malignant disease.
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Neoplasias de las Glándulas Suprarrenales/diagnóstico , Neoplasias de las Glándulas Suprarrenales/patología , Células Cromafines/metabolismo , Células Cromafines/patología , Cromograninas/metabolismo , Feocromocitoma/diagnóstico , Feocromocitoma/patología , Neoplasias de las Glándulas Suprarrenales/metabolismo , Humanos , Feocromocitoma/metabolismoRESUMEN
Pheochromocytomas are catecholamine-producing tumors which are generally benign, but which can also present as or develop into malignancy. Molecular pathways of malignant transformation remain poorly understood. Pheochromocytomas express various trophic peptides which may influence tumoral cell behavior. Here, we investigated the expression of trophic amidated peptides, including pituitary adenylate cyclase-activating polypeptide (PACAP), neuropeptide Y (NPY), and adrenomedullin (AM), and their receptors in benign and malignant pheochromocytomas in order to assess their potential role in chromaffin cell tumorigenesis and malignant transformation. PACAP, NPY, and AM are expressed in the majority of pheochromocytomas studied; NPY exhibiting the highest mRNA levels relative to reference genes. Although median gene expression or peptide levels were systematically lower in malignant compared to benign tumors, no statistically significant difference was found. Among all the receptors of these peptides that were analyzed, only the AM receptor RDC1 displayed a differential expression between benign and malignant pheochromocytomas. This receptor exhibited a fourfold higher expression in malignant than in benign tumors. AM and stromal cell-derived factor 1, which has also been described as a ligand for RDC1, increased the number of human pheochromocytoma cells in primary culture and exerted anti-apoptotic activity on rat pheochromocytoma PC12 cells. In addition, RDC1 gene silencing decreased the number of viable PC12 cells. This study shows the expression of several trophic peptides and their receptors in benign and malignant pheochromocytomas, and suggests that AM and its RDC1 receptor could be involved in chromaffin cell tumorigenesis through pro-survival effects. Therefore, AM and RDC1 may represent valuable targets for the treatment of malignant pheochromocytomas.
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Neoplasias de las Glándulas Suprarrenales/metabolismo , Feocromocitoma/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/metabolismo , Receptores de Péptidos/biosíntesis , Neoplasias de las Glándulas Suprarrenales/genética , Adrenomedulina/biosíntesis , Adrenomedulina/genética , Animales , Western Blotting , Supervivencia Celular/fisiología , Formazáns/química , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Neuropéptido Y/biosíntesis , Neuropéptido Y/genética , Células PC12 , Feocromocitoma/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Ratas , Receptores de Adrenomedulina , Receptores de Neuropéptido Y/biosíntesis , Receptores de Neuropéptido Y/genética , Receptores de Péptidos/genética , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/biosíntesis , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sales de Tetrazolio/químicaRESUMEN
Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N- and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N- and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.
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Proteínas Anfibias/biosíntesis , Cromogranina A/biosíntesis , Péptidos/metabolismo , Proopiomelanocortina/metabolismo , Proteínas Recombinantes/biosíntesis , Vesículas Secretoras/metabolismo , Proteínas Anfibias/genética , Animales , Anuros , Células COS , Chlorocebus aethiops , Cromogranina A/genética , Expresión Génica , Péptidos/genética , Proopiomelanocortina/biosíntesis , Proteínas Recombinantes/genética , Vesículas Secretoras/genéticaRESUMEN
UNLABELLED: The gastroprokinetic agent metoclopramide is known to stimulate catecholamine secretion from pheochromocytomas. The aim of the study was to investigate the mechanism of action of metoclopramide and expression of serotonin type 4 (5-HT(4)) receptors in pheochromocytoma tissues. Tissue explants, obtained from 18 pheochromocytomas including the tumor removed from a 46-year-old female patient who experienced life-threatening hypertension crisis after metoclopramide administration and 17 additional pheochromocytomas (9 benign and 8 malignant) were studied. Cultured pheochromocytoma cells derived from the patient who previously received metoclopramide were incubated with metoclopramide and various 5-HT(4) receptor ligands. In addition, total mRNAs were extracted from all the 18 tumors. Catecholamine- and granin-derived peptide concentrations were measured in pheochromocytoma cell incubation medium by HPLC and radioimmunological assays. In addition, expression of 5-HT(4) receptor mRNAs in the 18 pheochromocytomas was investigated by the use of reverse transcriptase-PCR. RESULTS: Metoclopramide and the 5-HT(4) receptor agonist cisapride were found to activate catecholamine- and granin-derived peptide secretions by cultured tumor cells. Metoclopramide- and cisapride-evoked catecholamine- and granin-derived peptide productions were inhibited by the 5-HT(4) receptor antagonist GR 113808. 5-HT(4) receptor mRNAs were detected in the patient's tumor and the series of 17 additional pheochromocytomas. This study shows that pheochromocytomas express functional 5-HT(4) receptors that are responsible for the stimulatory action of metoclopramide on catecholamine- and granin-derived peptide secretion. All 5-HT(4) receptor agonists must therefore be contraindicated in patients with proven or suspected pheochromocytoma.
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Neoplasias de las Glándulas Suprarrenales/tratamiento farmacológico , Antagonistas de Dopamina/farmacología , Metoclopramida/farmacología , Feocromocitoma/tratamiento farmacológico , Receptores de Serotonina 5-HT4/genética , Neoplasias de las Glándulas Suprarrenales/metabolismo , Médula Suprarrenal/citología , Médula Suprarrenal/efectos de los fármacos , Catecolaminas/metabolismo , Cromograninas/metabolismo , Cisaprida/farmacología , Contraindicaciones , Domperidona/farmacología , Femenino , Humanos , Persona de Mediana Edad , Feocromocitoma/metabolismo , ARN Mensajero/metabolismo , Receptores de Serotonina 5-HT4/metabolismo , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Agonistas de Receptores de Serotonina/farmacología , Células Tumorales CultivadasRESUMEN
The last 5 years have witnessed important advances in understanding the mechanisms of tumorigenesis of chromaffin cells. Large-scale microarray analyses of pheochromocytomas have identified two distinct gene-expression profiles encompassing all hereditary and sporadic tumors. Gene-expression profiling of benign and malignant pheochromocytomas is providing a better understanding of the mechanisms of metastasis. Such studies hold promise for the development of new prognostic markers for early detection of malignant pheochromocytoma and for the identification of novel targets for therapeutic intervention.
RESUMEN
An increase in circulating catecholamine levels represents one of the mechanisms whereby organisms cope with stress. In the periphery, catecholamines mainly originate from the sympathoadrenal system. As we reported, in addition to the central control through cholinergic innervation, a local gap junction-delineated route between adrenal chromaffin cells contributes to catecholamine exocytosis. Here, we investigated whether this intercellular communication is modified when the hormonal demand is increased as observed during cold stress. Our results show that in cold exposed rats, gap-junctional communication undergoes a functional plasticity, as evidenced by an increased number of dye-coupled cells. Of a physiological interest is that this upregulation of gap-junctional coupling results in the appearance of a robust electrical coupling between chromaffin cells that allows the transmission of action potentials between coupled cells. This enhancement of gap-junctional communication parallels an increase in expression levels of connexin36 (Cx36) and connexin43 (Cx43) proteins. Both transcriptional and posttranslational mechanisms are involved because Cx36 transcripts are increased in stressed rats and the expression of the scaffolding protein zonula occludens-1, known to interact with both Cx36 and Cx43, is also upregulated. Consistent with an upregulated coupling extent in stressed rats, the cytosolic Ca(2+) concentration rises triggered in a single cell by an iontophoretic application of nicotine occur simultaneously in several neighboring cells. These results describe for the first time a functional plasticity of junctional coupling between adult chromaffin cells that should be crucial for adaptation to stress or sensitization to subsequent stressors.
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Médula Suprarrenal/metabolismo , Catecolaminas/metabolismo , Comunicación Celular/fisiología , Células Cromafines/metabolismo , Uniones Comunicantes/metabolismo , Estrés Psicológico/metabolismo , Potenciales de Acción/fisiología , Médula Suprarrenal/ultraestructura , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Células Cromafines/ultraestructura , Frío/efectos adversos , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Uniones Comunicantes/ultraestructura , Masculino , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Plasticidad Neuronal/fisiología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Ratas , Ratas Wistar , Estrés Psicológico/fisiopatología , Regulación hacia Arriba/fisiología , Proteína de la Zonula Occludens-1 , Proteína delta-6 de Union ComunicanteRESUMEN
CONTEXT: Pheochromocytomas are catecholamine-producing tumors that are generally benign but that can also present as or develop into malignancy. Occurrence of malignant pheochromocytomas can only be asserted by imaging of metastatic lesions. OBJECTIVES: We conducted a gene expression profiling of benign and malignant tumors to identify a gene signature that would allow us to discriminate benign from malignant pheochromocytomas and to gain a better understanding of tumorigenic pathways associated with malignancy. DESIGN: A total of 36 patients with pheochromocytoma was studied retrospectively. There were 18 (nine benign and nine malignant) tumors used for gene expression profiling on pangenomic oligonucleotide microarrays. RESULTS: We identified and validated a set of predictor genes that could accurately distinguish the two tumor subtypes through unsupervised clustering. Most of the differentially expressed genes were down-regulated in malignant tumors, and several of these genes encoded neuroendocrine factors involved in prominent characteristics of chromaffin cell biology. In particular, the expression of two key processing enzymes of trophic peptides, peptidylglycine alpha-amidating monooxygenase and glutaminyl-peptide cyclotransferase, was reduced in malignant pheochromocytomas. CONCLUSION: The gene expression profiling of benign and malignant pheochromocytomas clearly identified a set of genes that could be used as a prognostic multi-marker and revealed that the expression of several genes encoding neuroendocrine proteins was reduced in malignant compared with benign tumors.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/patología , Feocromocitoma/genética , Feocromocitoma/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Femenino , Marcadores Genéticos , Humanos , Lactante , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
EM66 is a 66-amino acid peptide derived from secretogranin II, a member of granin acidic secretory protein family, by proteolytic processing. EM66 has been previously characterized in the jerboa (Jaculus orientalis) hypothalamus and its potential implication in the neuroendocrine regulation of feeding behaviour has been demonstrated. In the present study, an immunohistochemical analysis of the localization of EM66 within hypothalamic structures of rat was performed and compared to the distribution of EM66 in the jerboa hypothalamus. In the rat hypothalamus, as in the jerboa, EM66 immunostaining was detected in the parvocellular paraventricular, preoptic and arcuate nuclei, as well as the lateral hypothalamus which displayed an important density of EM66-producing neurones. However, unlike jerboa, the suprachiasmatic and supraoptic nuclei of the rat hypothalamus were devoid of cellular EM66-immunolabeling. Thus, the novel peptide EM66 may exert common neuroendocrine activities in rat and jerboa, e.g. control of food intake, and species-specific roles in jerboa such as the regulation of biological rhythms and hydromineral homeostasis. These results suggest the existence of differences between jerboas and rats in neuroendocrine regulatory mechanisms involving EM66.
Asunto(s)
Hipotálamo/metabolismo , Neuropéptidos/metabolismo , Fragmentos de Péptidos/metabolismo , Ratas Wistar/metabolismo , Roedores/metabolismo , Secretogranina II/metabolismo , Animales , Mapeo Encefálico , Conducta Alimentaria/fisiología , Femenino , Homeostasis/fisiología , Hipotálamo/anatomía & histología , Inmunohistoquímica , Masculino , Neuronas/metabolismo , Neuropéptidos/química , Sistemas Neurosecretores/metabolismo , Fragmentos de Péptidos/química , Periodicidad , Ratas , Ratas Wistar/anatomía & histología , Roedores/anatomía & histología , Secretogranina II/química , Especificidad de la Especie , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
The aim of the present study was to compare the expression levels of secretogranin II (SgII), prohormone convertases (PC)1 and PC2, and the proteolytic processing of SgII in benign versus malignant pheochromocytomas. Quantitative (Q)-PCR experiments indicated that SgII, PC1, and PC2 mRNAs were overexpressed in pheochromocytoma compared to non-tumoral chromaffin cells (P<0.001) and in benign compared to malignant tumors (P<0.01). Western blot analysis using a human SgII antiserum revealed the occurrence of a 97-kDa band corresponding to the expected size of SgII, with significantly higher quantities in benign than in malignant tumors (P<0.05). Using antisera directed against sequential regions of SgII (N-terminal, secretoneurin [SN], EM66, internal, and C-terminal sequences), we observed distinct processing profiles between benign and malignant pheochromocytomas. In contrast, using PC1 and PC2 antisera no differences between the two types of tumors were found. RIA measurement showed that EM66 median values between benign and malignant chromaffin cell tumors were significantly different (128.5 vs. 6.3 ng/mg protein, respectively; P<0.001). Taken together, these results indicate that, in pheochromocytoma, malignancy is associated with reduced PC1, PC2, and SgII mRNA expression and decreased levels of processing products of SgII, in line with the low concentrations of EM66 that occur in malignant tumors. These data support the notion that SgII-processing products, such as EM66, could represent prognostic markers of pheochromocytomas.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/diagnóstico , Perfilación de la Expresión Génica , Feocromocitoma/diagnóstico , Neoplasias de las Glándulas Suprarrenales/patología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Feocromocitoma/patología , Pronóstico , RadioinmunoensayoRESUMEN
Pheochromocytomas (PHEOs) are rare catecholamine-producing neoplasias that arise from chromaffin cells of the adrenal medulla or from extra-adrenal locations. These neuroendocrine tumors are usually benign, but may also present as or develop into a malignancy. There are currently no means to predict or to cure malignant tumors which have a poor prognosis. We have recently validated several assays for the measurement of peptides derived from chromogranin A (CgA) and secretogranin II (SgII) in order to determine whether these secreted neuroendocrine products could provide useful, complementary markers for the diagnosis and prognosis of PHEOs. Both the CgA-derived peptide WE14 and the SgII-derived peptide EM66 proved to be sensitive circulating markers for the diagnosis of PHEO. In addition, much higher EM66 levels were measured in benign than in malignant tumoral tissues, suggesting that this peptide could represent a valuable tool for the prognosis of PHEO. We have also initiated a comparative microarray study of benign and malignant PHEOs, which allowed the identification of a set of about 100 genes that were differentially expressed and best discriminated the two types of tumors. A large majority of these genes were expressed at lower levels in the malignant disease and were associated with various characteristics of chromaffin cells, such as hormone secretion signaling and machinery, peptide maturation, and cellular morphology. Altogether, these studies provide novel tools for the management of PHEO, and new insights for the understanding of tumorigenesis in chromaffin cells, which may offer potential therapeutic strategies.
Asunto(s)
Neoplasias de las Glándulas Suprarrenales/genética , Perfilación de la Expresión Génica , Feocromocitoma/genética , Humanos , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Secretoneurin (SN) and EM66 are two highly conserved peptides that derive from the processing of secretogranin II (SgII), one of the major constituents of chromaffin cell secretory vesicles. It has been shown that PACAP regulates SgII gene transcription and SN release in bovine adrenochromaffin cells. The aim of the present study was to localize and characterize EM66 in the bovine adrenal gland, and to examine the signaling pathways activated by PACAP to regulate the secretion of EM66 from cultured chromaffin cells. Double immunohistochemical labeling showed an intense EM66-immunoreactive (EM66-IR) signal in TH-positive medullary chromaffin cells of the adrenal gland. HPLC analysis combined with RIA detection revealed, in adrenal medulla extracts and cultured chromaffin cell media, the presence of a major EM66-IR peak co-eluting with the recombinant peptide. PACAP dose-dependently stimulated EM66 release from chromaffin cells (ED(50)=4.8 nM). The effect of PACAP on EM66 secretion was observed after 6 h of treatment and increased to reach a 2.6-fold stimulation at 48 h. The nonselective calcium channel blocker NiCl(2), the cytosolic calcium chelator BAPTA-AM and the L-type calcium channel blocker nimodipine significantly inhibited the stimulatory effect of PACAP on EM66 release. The secretory response to PACAP was also significantly lowered by the protein kinase A inhibitor H89 and by the protein kinase C inhibitor chelerythrine. Concomitant administration of chelerythrine, H89, NiCl(2) and BAPTA totally abolished PACAP-stimulated EM66 secretion. The MAPK inhibitors U0126 and SB203580 respectively decreased by 63% and 72% PACAP-evoked EM66 release. These results indicate that, in bovine adrenal medulla, SgII is processed to generate the EM66 peptide and that PACAP activates multiple signaling pathways to regulate EM66 release from chromaffin cells, suggesting that EM66 may act downstream of the trans-synaptic stimulation of the adrenal medulla by neurocrine factors.
Asunto(s)
Células Cromafines/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Transducción de Señal , Glándulas Suprarrenales/metabolismo , Animales , Western Blotting , Bovinos , Células Cromafines/metabolismo , Cromatografía Líquida de Alta Presión , Inmunohistoquímica , Técnicas In Vitro , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fragmentos de Péptidos/metabolismo , Radioinmunoensayo , Secretogranina II/metabolismoRESUMEN
We have previously shown that PACAP stimulates in vitro the secretion of corticosteroids by frog adrenal explants and that PACAP increases cAMP formation and cytosolic calcium concentration ('Ca2+'i) in adrenocortical cells. The aim of the present study was to investigate the involvement of cAMP and 'Ca2+'i in the stimulatory effect of PACAP on steroid production. Incubation of adrenal explants with PACAP resulted in a significant increase in total inositol phosphate formation. Administration of the protein kinase A inhibitor, H89, markedly reduced the stimulatory effect of PACAP on corticosterone and aldosterone secretion by perifused adrenal slices. In contrast, chelation of intracellular or extracellular calcium, or incubation with calcium channel blockers, had no effect on PACAP-evoked steroid secretion. Incubation of the cells with BAPTA or thapsigargin totally suppressed the stimulatory effect of PACAP on 'Ca2+'i. In contrast, suppression of extracellular calcium with EGTA or blockage of voltage-dependent Ca2+ channels did not impair PACAP-induced Ca2+ response. These data indicate that, in frog adrenocortical cells, the stimulatory effect of PACAP on steroid secretion is mediated through activation of the cAMP/PKA pathway. Concurrently, PACAP causes calcium mobilization from IP(3)-dependent intracellular stores through activation of a phospholipase C, while the calcium response is not involved in the stimulatory effect of PACAP on corticosteroid secretion.