Chromogranins as molecular coordinators at the crossroads between hormone aggregation and secretory granule biogenesis

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Chromogranins as molecular coordinators at the crossroads between hormone aggregation and secretory granule biogenesis

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Chromogranin A as a Crucial Factor in the Sorting of Peptide Hormones to Secretory Granules

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Advanced Imaging Approaches to Reveal Molecular Mechanisms Governing Neuroendocrine Secretion

International audienceIdentification of the molecular mechanisms governing neuroendocrine secretion and resulting intercellular communication is one of the great challenges of cell biology to better understand organism physiology and neurosecretion disruption-related pathologies such as hypertension, neurodegenerative, or metabolic diseases. To visualize molecule distribution and dynamics at the nanoscale, many imaging approaches have been developed and are still emerging. In this review, we provide an overview of the pioneering studies using transmission electron microscopy, atomic force microscopy, total internal reflection microscopy, and super-resolution microscopy in neuroendocrine cells to visualize molecular mechanisms driving neurosecretion processes, including exocytosis and associated fusion pores, endocytosis and associated recycling vesicles, and protein-protein or protein-lipid interactions. Furthermore, the potential and the challenges of these different advanced imaging approaches for application in the study of neuroendocrine cell biology are discussed, aiming to guide researchers to select the best approach for their specific purpose around the crucial but not yet fully understood neurosecretion process

Phosphatidic acid: Mono- and poly-unsaturated forms regulate distinct stages of neuroendocrine exocytosis

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Involvement of multiple signaling pathways in PACAP-induced EM66 secretion from chromaffin cells

International audienceSecretoneurin (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

PACAP Stimulates the Release of the Secretogranin II-Derived Peptide EM66 from Chromaffin Cells

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Biochemical Characterisation and Immunohistochemical Localisation of the Secretogranin II-Derived Peptide EM66 in the Hypothalamus of the Jerboa (Jaculus orientalis): Modulation by Food Deprivation

International audienceThe neuroendocrine protein secretogranin II is the precursor of several neuropeptides, including secreto-neurin and a novel 66-amino acid peptide, EM66, the sequence of which has been highly conserved across the vertebrae phylum. The presence of EM66 has been detected in the adult and fetal human adrenal gland, as well as the rat pituitary and adrenal glands. The present study aimed to explore a possible neuroendocrine role of EM66 by analysing its occurrence and distribution within the jerboa hypothalamus, and its potential implication in the control of feeding behaviour. High-performance liquid chromatography analysis of jerboa hypothalamic extracts combined with a radioimmunoassay of EM66 revealed a single peak of immunoreactive material exhibiting the same retention time as recombinant EM66. Immuno-cytochemical labelling showed that EM66-producing neurones are widely distributed in several hypotha-lamic regions, including the preoptic area, the suprachiasmatic, supraoptic, parvocellular paraventricular and arcuate nuclei, and the lateral hypothalamus. Food deprivation for 5 days induced a significant increase in the number of EM66-containing neurones within the arcuate nucleus (105% increase) and the parvo-cellular aspect of the paraventricular nucleus (115% increase), suggesting that EM66 could be involved in the control of feeding behaviour and/or the response to stress associated with fasting. Altogether, these data reveal the physiological plasticity of the EM66 system in the hypothalamus and implicate this novel peptide in the regulation of neuroendocrine functions

Characterization and Plasma Measurement of the WE-14 Peptide in Patients with Pheochromocytoma

International audienceGranins 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

Identification of the Secretogranin II-Derived Peptide EM66 in Pheochromocytomas as a Potential Marker for Discriminating Benign Versus Malignant Tumors

International audienceEM66 is a novel secretogranin II-derived peptide present in chromaffin cells of the human adrenal gland. The aim of the present study was to investigate the possible occurrence of EM66 in benign and malignant pheochromocytomas. Immu-nohistochemical labeling using specific antibodies revealed intense staining in both benign and malignant tumors. Coin-cubation of pheochromocytoma slices with EM66 and tyrosine hydroxylase antibodies showed that the immunostaining was restricted to chromaffin cells. RIA experiments indicated that serial dilutions of extracts of benign and malignant tumors generated displacement curves that were parallel to those produced by recombinant EM66. RIA quantification revealed concentrations of EM66 immunoreactivity ranging from 3.2– 210 ng/mg protein (median 25.6 ng/mg protein) in benign pheochromocytomas, and from 2.9 – 6.3 ng/mg protein (median 3.8 ng/mg protein) in malignant tumors. The EM66-like immunoreactivity contained in the pheochromocytoma extracts was characterized by HPLC analysis combined with RIA detection. All of the benign and malignant tumors examined exhibited a single immunoreactive peak coeluting with recombinant EM66. These data indicate that the secretogra-nin II-derived peptide EM66 is generated in human tumoral chromaffin tissue. The significant difference in EM66 concentrations observed between benign and malignant pheochro-mocytomas suggests that measurement of EM66 levels may help identifying patients with higher risk of progression of such tumors. (J Clin Endocrinol Metab 88: 2579 –2585, 2003