Laurent Yon

Chromogranin A Promotes Peptide Hormone Sorting to Mobile Granules in Constitutively and Regulated Secreting Cells ROLE OF CONSERVED N- AND C-TERMINAL PEPTIDES

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.

Neuroanatomical and Physiological Evidence for the Involvement of Pituitary Adenylate Cyclase-Activating Polypeptide in the Regulation of the Distal Lobe of the Frog Pituitary

Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a 38 amino‐acid peptide which belongs to the glucagon/secretin/ vasoactive intestinal peptide superfamily. The sequence of PACAP is identical in all mammalian species studied so far but frog PACAP differs by one amino‐acid from mammalian PACAP. The aim of the present study was to investigate the presence of PACAP in the hypothalamo‐pituitary complex of the frog Rana ridibunda and to determine the biological activity of frog PACAP on homologous pituitary cells. The distribution of PACAP‐containing neurons and fibers was examined by the indirect immunofluores‐cence method using an antiserum raised against the N‐terminal region of the peptide. In the hypothalamus, PACAP‐immunoreactive perikarya were localized in the preoptic nucleus and the dorsal and ventral infundibular nuclei. Beaded nerve fibers were observed coursing from the ventral infundibular nucleus to the external vascular layer of the median eminence. A dense network of immunoreactive axons terminated in the vicinity of the capillaries of the hypophysial portal system. The neurointermediate lobe and the distal lobe of the pituitary were devoid of immunoreactive elements. The amount of PACAP‐like immunoreactive material in hypothalamus extracts was measured by radioimmunoassay; the apparent concentration of PACAP was 4.5 ng/mg protein. Synthetic frog PACAP38 and PACAP27 induced a similar dose‐dependent stimulation of cAMP production in isolated frog distal lobe pituitary fragments (ED50= 2 × 10−8 M). At the maximum dose tested (5 × 10−6 M), both frog PACAP38 and PACAP27 produced a 4‐fold increase in cAMP production. In contrast, the truncated form [Des‐His1frog PACAP38 did not affect adenylate cyclase activity demonstrating therefore that the N‐terminal histidyl residue is essential for the biological activity of the peptide. [Des‐His1]frog PACAP38 did not antagonize the stimulatory effect of frog PACAP38 or PACAP27 on cAMP production. Taken together, these data support the concept that, in amphibians as in mammals, PACAP may act as a hypophysiotropic neuropeptide.

Biochemical Characterization and Immunocytochemical Localization of EM66, a Novel Peptide Derived from Secretogranin II, in the Rat Pituitary and Adrenal Glands

Characterization of secretogranin II (SgII) mRNA in various vertebrates has revealed selective conservation of the amino acid sequences of two regions of the protein, i.e., the bioactive peptide secretoneurin and a flanking novel peptide that we named EM66. To help elucidate the possible role of EM66, we examined the occurrence as well as the cellular and subcellular distribution of EM66 in rat pituitary and adrenal glands by using a polyclonal antibody raised against the recombinant human EM66 peptide. High-performance liquid chromatography (HPLC) analysis of rat pituitary and adrenal extracts combined with a radioimmunoassay resolved EM66-immunoreactive material exhibiting the same retention time as recombinant EM66. In the rat pituitary, double-labeling immunohistochemical (IHC) studies showed that EM66 immunoreactivity (IR) was present in gonadotrophs, lactotrophs, thyrotrophs, and melanotrophs, whereas corticotrophs were devoid of labeling. EM66-IR was also observed in nerve endings in the neural lobe. Immunocytochemical staining at the electron microscopic level revealed that EM66-IR is sequestered in the secretory granules within gonadotrophs and lactotrophs. In the adrenal medulla, double IHC labeling showed that EM66-IR occurs exclusively in epinephrine-synthesizing cells. At the ultrastructural level, EM66-IR was seen in chromaffin vesicles of adrenomedullary cells. These results demonstrate that post-translational processing of SgII generates a novel peptide that exhibits a cell-specific distribution in the rat pituitary and adrenal glands where it is stored in secretory granules, supporting the notion that EM66 may play a role in the endocrine system.

Functional Remodeling of Gap Junction-Mediated Electrical Communication between Adrenal Chromaffin Cells in Stressed Rats

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 Ca2+ 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.

Circulating EM66 is a highly sensitive marker for the diagnosis and follow-up of pheochromocytoma

We have previously demonstrated that measurement of tissue concentration of the novel secretogranin II‐derived peptide EM66 may help to discriminate between benign and malignant pheochromocytomas. The aim of the present study was to characterize EM66 in plasma and urine of healthy volunteers and pheochromocytoma patients, in order to further evaluate the usefulness of this peptide as a circulating marker for the management of the tumors. HPLC analysis of plasma and urine samples demonstrated that the EM66‐immunoreactive material coeluted with the recombinant peptide. In healthy volunteers, plasma and urinary EM66 levels were, respectively, 2.6 (1.9–3.7) ng/ml and 2.9 (1.9–4.6) ng/ml. In patients with pheochromocytoma, plasma EM66 levels were 10‐fold higher than those of healthy volunteers (26.9 (7.3–44) ng/ml), and returned to normal values after removal of the tumor. In contrast, urinary EM66 levels were not significantly different from those of healthy volunteers (3.2 (2.2–3.9) ng/ml). Measurement of total or free plasma metanephrines and 24 hr urinary metanephrines in our series of patients revealed that these tests, taken separately, are less sensitive than the EM66 determination. Pheochromocytes in primary culture secreted high levels of EM66, suggesting that the chromaffin tumor was actually responsible for the increased plasma peptide concentrations in the patients. These data indicate that EM66 is secreted in the general circulation and that elevated plasma EM66 levels are correlated with the occurrence of pheochromocytoma. Thus, EM66 is a sensitive plasma marker that should be considered as a complementary tool in the management of pheochromocytoma.

Development of Novel Tools for the Diagnosis and Prognosis of Pheochromocytoma Using Peptide Marker Immunoassay and Gene Expression Profiling Approaches

Abstract:  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.

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

The neuroendocrine protein secretogranin II is the precursor of several neuropeptides, including secretoneurin 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. Immunocytochemical labelling showed that EM66‐producing neurones are widely distributed in several hypothalamic 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 parvocellular 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 localization of pituitary adenylate cyclase-activating polypeptide (PACAP) binding sites in the brain of the frog Rana ridibunda.

The biochemical characteristics and the distribution of pituitary adenylate cyclase–activating polypeptide (PACAP) binding sites have been investigated in the brain of the frog Rana ridibunda by using [125I]PACAP27 as a radioligand. Membrane‐binding studies revealed the existence of high‐affinity receptors for frog PACAP38 and PACAP27. In contrast, the [Des‐His1]PACAP38 analogue had a much lower affinity and vasoactive intestinal polypeptide did not produce any displacement of the binding. Autoradiographic labeling of frozen brain sections revealed that the highest concentrations of PACAP receptors were located in the olfactory bulb, pallium, striatum, habenular nuclei, ventromedial thalamic nucleus, corpus geniculatum, posterior tubercle, dorsal part of the magnocellular preoptic nucleus, tectum, and the molecular cell layer of the cerebellum. Moderate binding was observed in the septum, in most parts of the thalamus, the dorsal hypothalamic nucleus, the median eminence, the ventral nuclei of the tegmentum, the torus semicircularis, and the interpeduncular and isthmi nuclei. The present data provide the first biochemical characterization and anatomic distribution of PACAP binding sites in the brain of a nonmammalian vertebrate species. The widespread distribution of specific PACAP receptors in the frog brain suggests that the peptide does not act solely as a hypophysiotropic factor, but likely fulfills neurotransmitter functions, neuromodulator functions, or both. J. Comp. Neurol. 412:218–228, 1999.

Granins and their derived peptides in normal and tumoral chromaffin tissue: Implications for the diagnosis and prognosis of pheochromocytoma

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.

Normotensive Incidentally Discovered Pheochromocytomas Display Specific Biochemical, Cellular, and Molecular Characteristics

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.