Maité Montero-Hadjadje

Georges Pelletier

Laurent Yon

Songyun Li

Johann Guillemot

Rabia Magoul

Yves Tillet

Hubert Vaudry
email: hubert.vaudry@univ-rouen.fr

Youssef Anouar

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

A R Y 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. (

Secretogranin II (SgII) belongs to the chromogranin/secretogranin protein family whose members occur in most endocrine, neuroendocrine, and nerve cells [START_REF] Rosa | The granin protein family: markers for neuroendocrine cells and tools for the diagnosis of neuroendocrine tumors[END_REF][START_REF] Fischer-Colbrie | Secretogranin II: molecular properties, regulation of biosynthesis and processing to the neuropeptide secretoneurin[END_REF]. These acidic secretory proteins play a role in the morphogenesis of secretory vesicles [START_REF] Iacangelo | Chromogranin A: current status as a precursor for bioactive peptides and a granulogenic/sorting factor in the regulated secretory pathway[END_REF][START_REF] Kim | Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis[END_REF]. There is also evidence that granins are precursor proteins that can be processed by prohormone convertases (PCs) to generate bioactive peptides [START_REF] Simon | Biochemistry of the chromogranin A protein family[END_REF][START_REF] Winkler | The chromogranins A and B: the first 25 years and future perspectives[END_REF][START_REF] Iacangelo | Chromogranin A: current status as a precursor for bioactive peptides and a granulogenic/sorting factor in the regulated secretory pathway[END_REF]. In particular, SgII is the precursor of a peptide named secretoneurin (SN) [START_REF] Vaudry | Identification of a peptide arising from the specific post-translation processing of secretogranin II[END_REF][START_REF] Kirchmair | Secretoneurin-a neuropeptide generated in brain, adrenal medulla and other endocrine tissues by proteolytic processing of secretogranin II (chromogranin C)[END_REF]) which has been implicated in the modulation of the inflammatory response and may represent a novel sensory peptide for neuroimmunomodulation [START_REF] Wiedermann | Secretoneurin: a functional neuropeptide in health and disease[END_REF]. The cloning of the cDNAs encoding various members of the chromogranin/secretogranin family in species that are phylogenetically distant from mammals, has revealed the existence within these proteins of a few domains that have been remarkably conserved during evolution, whereas the remaining sequences profoundly diverged among species (Anouar et al. 1996;[START_REF] Holthuis | The neuroendocrine proteins secretogranin II and III are regionally conserved and coordinately expressed with proopiomelanocortin in Xenopus intermediate pituitary[END_REF]Blazquez et al. 1998;[START_REF] Turquier | Frog chromogranin A messenger ribonucleic acid encodes three highly conserved peptides. Coordinate regulation of proopiomelanocortin and chromogranin A gene expression in the pars intermedia of the pituitary during background color adaptation[END_REF]Ait-Ali et al. 2002). Moreover, the sequences of the conserved regions are delimited by preserved dibasic amino acids which represent potential cleavage sites for PCs [START_REF] Laslop | Proteolytic processing of chromogranins[END_REF][START_REF] Turquier | Characterization of chromogranins in the frog Rana ridibunda: structure, expression, and functional implications[END_REF]. For example, although SgII displays only 46% overall amino acid sequence identity between frog and human, high sequence homology occurs in the regions encoding SN and its C-terminal flanking peptide, and the two sequences are bounded by paired basic residues (Figure 1) (Anouar et al. 1996;[START_REF] Holthuis | The neuroendocrine proteins secretogranin II and III are regionally conserved and coordinately expressed with proopiomelanocortin in Xenopus intermediate pituitary[END_REF]Blazquez et al. 1998). This observation suggests that the 66-amino-acid peptide flanking SN may also give rise to a mature peptide in endocrine, neuroendocrine, and neuronal cells that could play regulatory functions. This peptide was named EM66 in consideration of its N-and C-terminal glutamic acid and methionine residues, respectively, in the human sequence. We have raised polyclonal antibodies against a fusion protein encompassing the human EM66 sequence that was produced in Escherichia coli. Using these antibodies, we have shown that EM66 is actually generated in the adult and fetal human adrenal gland, thus establishing the existence of this novel SgII-derived peptide (Anouar et al. 1998).

As a further step towards the elucidation of the possible role of EM66, the aim of the present study was to investigate the occurrence of EM66 in the rat pituitary and adrenal glands, where high amounts of the precursor protein SgII and its processing products are found (Anouar and Duval 1991;[START_REF] Lloyd | Chromogranin A, chromogranin B and secretogranin II mRNAs in the pitu-itary and adrenal glands of various mammals. Regulation of chromogranin A, chromogranin B and secretogranin II mRNA levels by estrogen[END_REF][START_REF] Fischer-Colbrie | Secretogranin II: molecular properties, regulation of biosynthesis and processing to the neuropeptide secretoneurin[END_REF][START_REF] Leitner | Secretogranin II: relative amounts and processing to secretoneurin in various rat tissues[END_REF], and to determinate the precise cellular and subcellular localization of EM66 within these tissues.

Materials and Methods

Animals and Tissue Samples

Three-month-old male Wistar rats weighing 250 to 300 g were maintained under controlled conditions of temperature (22C) and an established photoperiod (lights on from 0700 hr to 1900 hr). Rats had free access to laboratory chow (UAR; Epinay-sur-Orge, France) and water. All manipulations were performed according to the recommendations of the French Ethical Committee and under the supervision of authorized investigators. The animals were sacrificed by decapitation between 0830 hr and 0930 hr, and the adrenal glands and pituitaries were quickly removed and frozen at Ϫ80C.

Production of Recombinant EM66

A fusion protein containing the human EM66 peptide was produced in E. coli as previously described (Anouar et al. 1998). Briefly, the cDNA sequence encoding the EM66 region in human SgII was amplified by PCR, inserted downstream of the maltose-binding protein (MBP) region of the plasmid pMAL-c2 (New England Biolabs; Beverly, MA), and expressed in bacteria. The cleavage of the affinity-purified fusion protein with factor Xa releases EM66 with the native amino acid sequence.

Antibodies

Antibodies against EM66 (code 736-1806) were raised in rabbits against the recombinant fusion protein MBP-EM66, and their specificity has been established by Western blotting and IHC (Anouar et al. 1998). A mouse monoclonal antibody (code 518 B7) against the luteinizing hormone ␤-subunit (LH␤) was a generous gift from Dr J.F. Roser [START_REF] Matteri | Characterization of a monoclonal antibody which detects luteinizing hormone from diverse mammalian species[END_REF]. The adrenocorticotropin (ACTH) antibody (code 105-10) was raised against ACTH 1-24 in sheep and was provided by Dr J. Cote [START_REF] Jegou | Immunological characterization of endorphins, adrenocorticotropin, and melanotropins in frog hypothalamus[END_REF]. The development of the rabbit antibodies against rat prolactin (PRL) (code 19602), bovine phenylethanolamine N-methyltransferase (PNMT) (code 19886) and human thyroid-stimulating hormone ␤-subunit (TSH␤) has been previously described [START_REF] Martinat | Purification of rat prolactin: development of an homologous radioimmunological assay and comparison with the NIAMDD system[END_REF][START_REF] Tillet | Adrenergic neurons in sheep brain demonstrated by immunohistochemistry with antibodies to phenylethanolamine N-methyltransferase (PNMT) and dopamine-beta-hydroxylase (DBH): absence of the C1 cell group in the sheep brain[END_REF][START_REF] Uchiyama | Occurrence of immunoreactive activin/inhibin beta(B) in thyrotropes and gonadotropes in the bullfrog pituitary: possible paracrine/autocrine effects of activin B on gonadotropin secretion[END_REF]. The mouse monoclonal antibody directed against tyrosine hydroxylase (TH) was purchased from Chemicon International (Temecula, CA). Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse (GAM/FITC), FITC-conjugated donkey anti-sheep (DAS/FITC), FITC-conjugated goat anti-rabbit (GAR/FITC), and Texas Red-conjugated donkey anti-rabbit (DAR/TXR) ␥-globulins were from Amersham International (Poole, UK).

Immunohistochemical Procedure

For light microscopic IHC the rat adrenal and pituitary glands were dipped overnight in Stefanini's fixative consisting of 4% paraformaldehyde, 0.2% picric acid in 0.1 M phosphate buffer (PB), pH 7.4, rinsed with PB, and frozen at -80C in Tissue-Tek (Leica; Nussloch, Germany). Frozen glands were sliced into 10-m sections using a cryomicrotome (Frigocut; Reicher-Jung, Nussloch, Germany). After several rinses in PB, tissue sections were processed for indirect immunofluorescence microscopy. Slices were incubated overnight at 4C with one or two (in the case of double labeling) primary antibodies, i.e., the EM66 antiserum diluted 1:500, the TH antibody diluted 1:400, the LH␤ antibody diluted 1:300, the ACTH antiserum diluted 1:500, the PRL antiserum diluted 1:2000, the TSH␤ antiserum diluted 1:100, or the PNMT antiserum diluted 1:500 in PB containing 0.3% Triton X-100 and 1% bovine serum albumin (BSA; Roche Diagnostics, Mannheim, Germany). Tissue sections were rinsed in PB for 30 min and incubated for 90 min at room temperature (RT) with the appropriate secondary antibody, i.e., GAR/FITC, DAS/FITC, GAM/FITC diluted 1:100, or DAR/TXR diluted 1:50. Finally, slices were rinsed and mounted with PB/glycerol (1:1), coverslipped, and examined using a confocal laser scanning microscope (CLSM; Leica, Heidelberg, Germany) equipped with a diaplan optical system and an argon/krypton ion laser (excitation wavelengths 488/568/647 nm). To verify the specificity of the immunoreaction, various controls were performed: (a) substitution of the primary antisera/antibodies with PB; (b) incubation with non-immune serum instead of the primary antisera; (c) liquid-phase preabsorption of the EM66 antiserum (diluted 1:500) with purified recombinant peptide (10 Ϫ6 M).

Immunoelectron Microscopy

Three-month-old adult male Sprague-Dawley rats (225-250 g) were perfused transcardially with 200 ml of 2.5% glutaraldehyde in 0.1 M PB. Pituitaries and adrenal glands were excised and postfixed in the same fixative for 6 hr at 4C. Then the anterior lobes of the pituitaries and the adrenals were cut into small fragments 1ف( mm 3 ). The tissues were rinsed overnight in the same buffer and postfixed for 1 hr in 0.2% OsO 4 . After rinsing for 2 hr in distilled water, the tissues were dehydrated in ethanol and embedded in Araldite. Semithin sections were first performed for selection of the areas of interest in each gland. Ultrathin sections from the anterior lobe of the pituitary, and medullary and cortical zones of the adrenal gland, were then cut with a diamond knife and collected on nickel grids. Tissue sections were immunostained using the protein A-gold complex (10 nm; British Biocell, Cardiff, UK), as described [START_REF] Roth | Ultrastructural localization of intracellular antigens by the use of protein-A gold complex[END_REF]. The EM66 antiserum was used at a 1:200 dilution. Control experiments were performed by substituting non-immunized rabbit serum (1:200) or the antiserum (1:200) preabsorbed with an excess of antigen (10 Ϫ6 M). After the immunostaining procedures, the sections were counterstained with 1% uranyl acetate and lead citrate.

Tissue Extraction

Frozen rat pituitary and adrenal glands were immersed for 10 min in a cooled solution of 75% ethanol/18% hydrochloric acid (10 N). The tissue samples were sonicated and centrifuged at 10,000 ϫ g for 30 min at 4C. The supernatants were collected and dried by vacuum centrifugation (Speed-Vac Concentrator; Savant AES 2000, Hicksville, NY). The samples were reconstituted in 0.1% TFA in water and loaded onto three Sep-Pak C 18 cartridges (Waters Associates; Milford, MA) connected in series. The bound material was eluted with a solution of acetonitrile/water/TFA (49.9:50:0.1, v/v/v). The solvent was evaporated in the Speed-Vac Concentrator and the samples were kept dry until chromatographic analysis.

High-performance Liquid Chromatography (HPLC) Analysis

Dried samples were reconstituted in 1 ml of 0.1% TFA and centrifuged at 10,000 ϫ g for 10 min at 4C. The superna-tants were injected onto a 0.45 ϫ 25-cm Vydac C 18 column equilibrated with a solution of acetonitrile/water/TFA (9.9:90:0.1, v/v/v) at a flow rate of 1 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 60% over 25 min using a linear gradient. HPLC standard consisted of 1 g purified recombinant EM66. Fractions of 1 ml were collected, evaporated, and kept dry until RIA.

Radioimmunoassay

The concentrations of EM66-like material in the rat tissues were measured by RIA. Purified recombinant EM66 was iodinated by the chloramine-T method and separated from free iodine on Sep-Pak C 18 cartridges using a gradient of acetonitrile (0-100%) in 0.1% TFA. Radioiodinated EM66 eluted at 32% acetonitrile. The RIA was performed in 20 mM veronal buffer, pH 9.1, containing 3% BSA. The EM66 antiserum, used at a final dilution of 1:60,000, was incubated with 7000 cpm of tracer/tube in the presence of recombinant EM66 (standard peptide) or HPLC fractions. After a 2-day incubation at 4C, the antibody-bound fraction was immunoprecipitated by addition of 200 l goat anti-rabbit ␥-globulins (1:30), 200 l normal rabbit serum (1:150), and 500 l of 5% polyethyleneglycol 8000. After a 2-hr incubation at RT, the mixture was centrifuged (5000 ϫ g, 4C, 30 min), and the pellet containing the bound fraction was counted on a ␥-counter (LKB; Wallack, Rockville, MD).

Results

Characterization of EM66-IR in Pituitary and Adrenal Gland Extracts

Reversed-phase (RP)-HPLC analysis combined with RIA detection was conducted to characterize EM66 in rat pituitary and adrenal extracts (Figure 2). Recombi-nant EM66 eluted as a single peak with a retention time of 17.5 min (Figure 2A). A major EM66-IR peak exhibiting the same retention time as the recombinant peptide was detected in the adenohypophysial (Figure 2B) and adrenal (Figure 2C) gland extracts. A more hydrophobic EM66-immunoreactive compound eluting at 22.5 min was also resolved in the adenohypophysial gland extract (Figure 2B).

Distribution of EM66-IR in the Pituitary

The EM66 antiserum strongly labeled many cells in the anterior lobe (Figures 3A and3C) and moderately stained most melanotrophs in the intermediate lobe of the rat pituitary (Figures 3C and3D). At a higher magnification, EM66-IR was also observed in nerve endings in the neural lobe (Figure 3D). No staining was seen when the EM66 antiserum was preabsorbed with 10 Ϫ6 M of the recombinant peptide and when the primary antibody was substituted with non-immune rabbit serum or PB, thus confirming the specificity of the immunoreaction (Figure 3B).

Co-incubation of pituitary sections with the anti-EM66 antiserum and the anti-LH␤ antibody revealed that virtually all gonadotrophs contain EM66-IR (Fig- ures 4A-4C). However, some EM66-IR cells did not exhibit any LH␤-IR (Figures 4A-4C), indicating that the peptide occurs in cells other than gonadotrophs. Incubation of consecutive sections of the adenohypophysis with the anti-EM66 (Figure 5A) and anti-PRL (Figure 5B) antisera showed that some EM66-IR cells were lactotrophs (Figures 5A and5B). Similarly, incubation of consecutive sections with the anti-EM66 (Figure 6A) and the anti-TSH␤ (Figure 6B) antibodies revealed that EM66-IR is present in thyrotrophs (Figures 6A and6B). On the contrary, corticotrophs that were immunostained with the ACTH antibodies (Figure 7B) were totally devoid of EM66-IR (Figures 7A-7C).

The different types of pituitary cells can readily be identified on the basis of their ultrastructural characteristics [START_REF] Kurosumi | Functional classification of cell types in the anterior pituitary gland accomplished by electron microscopy[END_REF][START_REF] Pelletier | The secretory process in the anterior hypothesis[END_REF]. Somatotrophs are characterized by the presence of a large number of round or slightly ovoid secretory granules with a diameter of about 300-400 nm. Lactotrophs contain the largest granules of any pituitary cell. The mature secretory granules are often ovoid or elliptical, with a diameter ranging from 600 to 900 nm. Gonadotrophs are characterized by the presence of two types of gran- ules: the small (200 nm in diameter) and the large ones (350-700 nm in diameter). Corticotrophs can be identified by the presence of a single row of secretory granules (200 nm in diameter) exclusively located along the plasma membrane. Thyrotrophs have the smallest secretory granules (about 140 nm in diameter) of any cell type. EM66-IR material was only observed in secretory granules in gonadotrophs and lactotrophs. In gonadotrophs, both large and small secretory granules were decorated with gold particles (Figure 8A). The labeling of secretory granules of lactotrophs was generally weaker than that observed in gonadotrophs (Figure 8B). No labeling was observed in secretory granules of somatotrophs (Figure 8A). When the antiserum was absorbed with the antigen, no association of gold particles with any cell types in the anterior pituitary could be observed (data not shown).

Distribution of EM66-IR in the Adrenal Gland

The EM66 antibody strongly labeled the whole medulla (Figure 9A) and groups of cells located in the cortex (Figures 9A and9B) of the rat adrenal gland. Preabsorption of the primary antiserum with 10 Ϫ6 M EM66 totally abolished the labeling (data not shown). Co-incubation of adrenal sections with the anti-EM66 and anti-TH antibodies revealed that virtually all adrenomedullary cells that were immunolabeled with the EM66 antiserum were also stained with the TH antibodies (Figures 10A-10C), indicating that EM66 is present in chromaffin cells. However, a few THpositive cells were devoid of EM66-IR (Figure 10C). Double immunolabeling of adrenal slices with the anti-PNMT and anti-TH antibodies (Figures 10D-10F) revealed that the EM66-negative chromaffin cells were also PNMT-negative (Figures 10C and10F), indicat- ing that EM66 is present only in adrenergic cells and not in noradrenergic cells of the rat adrenal gland.

Immunocytochemical labeling of chromaffin cells showed that EM66-IR was restricted to secretory granules (Figures 11A and11B). In contrast, adrenocortical cells did not exhibit any staining (Figure 11A). When the antiserum was absorbed with the antigen, no association of gold particles with any cell types in the adrenal gland could be observed (data not shown).

Discussion

The present study has demonstrated the occurrence of a novel SgII-derived peptide, EM66, in the rat pituitary and adrenal, and has described its cellular and subcellular distribution in these endocrine glands.

Several lines of evidence indicate that SgII is a precursor protein that gives rise to biologically active peptides. (a) SgII contains pairs of basic amino acids that are known to be privileged targets of PCs in endocrine and neuroendocrine cells [START_REF] Gerdes | The primary structure of rat secretogranin II deduced from a cDNA sequence[END_REF][START_REF] Fischer-Colbrie | Sequence analysis, tissue distribution and regulation by cell depolarization, and second messengers of bovine secretogranin II (chromogranin C) mRNA[END_REF]Anouar et al. 1996). (b) SgII is cleaved by the convertases PC1 and PC2 [START_REF] Tooze | Antibodies to secretogranin II reveal potential processing sites[END_REF][START_REF] Laslop | Proteolytic processing of chromogranin B and secretogranin II by prohormone convertases[END_REF]). (c) A post-translational processing product of SgII, the peptide SN, has been identified in the brain of frog and mammals [START_REF] Vaudry | Identification of a peptide arising from the specific post-translation processing of secretogranin II[END_REF][START_REF] Marksteiner | Distribution of secretoneurinlike immunoreactivity in comparison with substance P-and enkephalin-like immunoreactivities in various human forebrain regions[END_REF]). (d) SN is released from endocrine and nerve cells upon stimulation [START_REF] Kirchmair | Human and rat primary C-fibre afferents store and release secretoneurin, a novel neuropeptide[END_REF][START_REF] Turquier | Pituitary adenylate cyclase-activating polypeptide stimulates secretoneurin release and secretogranin II gene transcription in bovine adrenochromaffin cells through multiple signaling pathways and increased binding of pre-exist-ing activator protein-1-like transcription factors[END_REF]. We have previously shown that SgII, in addition to SN, can give rise to the peptide EM66 in human adrenal gland (Anouar et al. 1998). Because the sequence of EM66 is highly conserved among species (Anouar et al. 1996), we postulated that the antisera directed against the human peptide may allow more precise characterization of the occurrence and distribution of EM66 in different endocrine tissues of rat. Analysis of tissue extracts by reversed-phase HPLC combined with RIA has revealed that EM66 is formed in the rat pituitary and adrenal, indicating that this SgII-derived peptide can be generated in various tissues and in different species. In the anterior pituitary, a second peak of EM66-IR material was also resolved. Owing to its higher retention time, this peak may correspond to an intermediate processing product of SgII, i.e., a C-and/or N-extended form of EM66.

In the anterior pituitary, intense EM66-IR was observed in all gonadotrophs as revealed by double labeling using a monoclonal antibody against LH␤. This finding is consistent with previous reports showing that SgII is a major product of gonadotrophs, in which its biosynthesis and release are regulated by gonadotropin-releasing hormone and sex steroids [START_REF] Chanat | The gonadotrope polypeptide (GP 87) released from pituitary cells under luteinizing hormone-releasing hormone stimulation is a secretogranin II form[END_REF]Anouar et al. 1991;Anouar and Duval 1992;[START_REF] Lloyd | Chromogranin A, chromogranin B and secretogranin II mRNAs in the pitu-itary and adrenal glands of various mammals. Regulation of chromogranin A, chromogranin B and secretogranin II mRNA levels by estrogen[END_REF][START_REF] Fischer-Colbrie | Secretogranin II: molecular properties, regulation of biosynthesis and processing to the neuropeptide secretoneurin[END_REF][START_REF] Watanabe | Effects of sex steroids on secretory granule formation in gonadotropes of castrated male rats with respect to granin expression[END_REF]). The intense EM66-IR observed in all gonadotrophs suggests that this peptide may represent a sensitive circulating marker for the diagnosis of human gonadotrope adenomas. In particular, EM66 may represent a valuable tool to characterize non-functioning pituitary tumors whose secretory granules have been shown to store SgII in the absence of the resident hormones [START_REF] Rosa | Widespread occurrence of chromogranins/secretogranins in the matrix of secretory granules of endocrinologically silent pituitary adenomas[END_REF][START_REF] Vallet | Secretogranin II (SgII) distribution and processing studies in human normal and adenomatous anterior pituitaries using new polyclonal antibodies[END_REF].

Immunofluorescence labeling and co-localization experiments revealed that EM66-IR could also be detected in few lactotrophs and thyrotrophs, but not in corticotrophs. Given that corticotrophs have been shown to express SgII mRNA [START_REF] Lloyd | Chromogranin A, chromogranin B and secretogranin II mRNAs in the pitu-itary and adrenal glands of various mammals. Regulation of chromogranin A, chromogranin B and secretogranin II mRNA levels by estrogen[END_REF], the lack of EM66 labeling suggests that either the SgII protein is not expressed or the mature EM66 peptide is not produced in these cells, at least at detectable levels using our antibodies. In addition, electron microscopic analysis showed that somatotrophs are also devoid of EM66-IR. The differential occurrence of EM66-IR may reflect cell-specific processing of SgII that could be ascribed to the efficiency with which PCs can act on the cleavage sites of EM66 in different pituitary cells. Thus, it is now well established that both PC1 and PC2 are expressed in several cell types of the anterior pituitary, including gonadotrophs [START_REF] Marcinkiewicz | Ontogeny of the prohormone convertases PC1 and PC2 in the mouse hypophysis and their colocalization with corticotropin and alpha-melanotropin[END_REF], corticotrophs [START_REF] Day | Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary[END_REF], and lactotrophs [START_REF] Muller | Immunocytochemical localization of the prohormone convertases PC1 and PC2 in rat prolactin cells[END_REF], and it has been shown that these processing enzymes are colocalized with chromogranins/secretogranins in pituitary and adrenal secretory granules [START_REF] Scopsi | Proprotein convertases (PC1/PC3 and PC2) in normal and neoplastic human tissues: their use as markers of neuroendocrine differentiation[END_REF]. However, the expression level and the activity of each PC may vary depending on the cell type [START_REF] Day | Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary[END_REF][START_REF] Marcinkiewicz | Ontogeny of the prohormone convertases PC1 and PC2 in the mouse hypophysis and their colocalization with corticotropin and alpha-melanotropin[END_REF], probably leading to cell-specific processing of SgII. Further studies using different cell lines transfected with SgII and PCs will be necessary to address this issue and to determine the efficiency of cleavage of EM66 from SgII. However, it should be noted that the HPLC analysis and the differential IHC localization of EM66 in the pituitary cells strongly suggest that this peptide is cleaved with a high efficiency and is the major immunoreactive SgIIderived form recognized by our antibody.

The present data also provide novel information regarding the cellular localization of a SgII-derived peptide in the rat adrenal medulla. Double labeling using antibodies against EM66, TH, and PNMT revealed that EM66-IR is restricted to a subset of TH-immunopositive cells, i.e., PNMT-expressing adrenergic cells. This finding highlights again the cell-specific occurrence of EM66 within endocrine tissues and implies either that SgII is expressed only in adrenergic cells, or that different mechanisms of SgII processing occur in adrenergic and noradrenergic chromaffin cells in vivo leading to production of EM66 in the former but not in the latter cells. Several studies using different experimental systems have shown that SgII can be cleaved by PC1 and PC2 in rat pheochromocytoma PC12 cells to give rise to several fragments including SN [START_REF] Dittie | Characterization of the endopeptidase PC2 activity towards secretogranin II in stably transfected PC12 cells[END_REF][START_REF] Hoflehner | Processing of secretogranin II by prohormone convertases: importance of PC1 in generation of secretoneurin[END_REF][START_REF] Laslop | Proteolytic processing of chromogranin B and secretogranin II by prohormone convertases[END_REF]). In addition, the presence of PC1 and PC2 has been described in chromaffin granules of the adrenal medulla [START_REF] Kirchmair | Secretoneurin-a neuropeptide generated in brain, adrenal medulla and other endocrine tissues by proteolytic processing of secretogranin II (chromogranin C)[END_REF]). However, it remains to be shown whether these enzymes are able to generate EM66 in chromaffin cells and whether the absence or presence of EM66-IR in chromaffin cell subpopulations is related to differential expression of PC1 and PC2 in these cells. Several reports have previously shown the existence of rays and islets of chromaffin cells in the adrenal cortex [START_REF] Palacios | Chromaffin cells in the glomerular zone of adult rat adrenal cortex[END_REF][START_REF] Gallo-Payet | On the presence of chromaffin cells in the adrenal cortex: their possible role in adrenocortical function[END_REF]Bornstein et al. 1991;[START_REF] Perraudin | Vasopressin stimulates cortisol secretion from human adrenocortical tissue through activation of V 1 receptors[END_REF]Berka et al. 1996). The present data indicate that these extramedullary adrenochromaffin cells also contain EM66-IR, suggesting that EM66 could play a role in the paracrine communication that occurs between chromaffin and steroidogenic cells in the adrenal gland (Bornstein and Ehrhart-Bornstein 1992;[START_REF] Ehrhart-Bornstein | Intraadrenal interactions in the regulation of adrenocortical steroidogenesis[END_REF]Bornstein et al. 2000;[START_REF] Delarue | Role of neurotransmitters and neuropeptides in the regulation of the adrenal cortex[END_REF].

Electron microscopic immunocytochemistry revealed that EM66-IR is confined to secretory vesicles of anterior pituitary and adrenomedullary chromaffin cells. Together with the HPLC data showing that mature EM66 peptide is present in the pituitary and the adrenal gland, these results imply that EM66 is formed into secretory granules. Consequently, it is conceivable that EM66 can be released into the external milieu by anterior pituitary cells and adrenomedullary cells on stimulation. This hypothesis raises the possibility that EM66 may exert hormonal activities. In fact, several observations suggest that EM66 may play endocrine functions. (a) The sequence of this peptide has been highly conserved during evolution and we have found that EM66 is actually processed in human (Anouar et al. 1998), rat (this study), and frog (Yon et al. unpublished work) endocrine glands. (b) EM66 is generated early during the ontogenesis of the fetal human adrenal gland (Anouar et al. 1998), suggesting that its formation is necessary throughout life. (c) EM66 exhibits a cell-specific occurrence in endocrine tissues, where it is confined to secretory vesicles. Therefore, EM66 may be released on stimulation of endocrine cells to act as an autocrine, paracrine, and/or endocrine factor. Alternatively, EM66 may represent a conserved structural motif allowing SgII to play an intracellular role in the morphogenesis and the function of secretory vesicles because the chromogranin/secretogranin family members are postulated to have a crucial role in these processes [START_REF] Yoo | Coupling of the inositol 1,4,5-trisphosphate receptor and chromogranins A and B in secretory granules[END_REF][START_REF] Kim | Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis[END_REF]. Further studies are required to test these hypotheses.

In conclusion, this study has demonstrated the occurrence of EM66, a novel peptide derived from the processing of SgII, in different cell types of the rat anterior pituitary and in adrenal chromaffin cells. In addition, the present data suggest that EM66 could be stored and released from the secretory granules of these cells and thus support the notion that EM66 may play a role in the endocrine system.

Literature Cited

Figure 1

 1 Figure 1 Schematic representation of the structure of SgII showing the location of conserved regions (shaded zones) between the frog and human SgII sequences. Two of these domains correspond to SN and EM66. The conserved dibasic sites and the percentages of amino acid identity between frog and human sequences are indicated.

Figure 2

 2 Figure2RP-HPLC analysis of EM66-like immunoreactivity (LI) in the rat pituitary and adrenal glands. (A) Elution profile of recombinant EM66 chromatographed onto a Vydac C 18 column. Fractions of 1 ml were collected, dried, and radioimmunoassayed for EM66. Protein extracts prepared from 50 pituitary anterior lobes (B) and from 30 adrenal glands (C) were pre-purified on Sep-Pak cartridges, analyzed as described above, and radioimmunoassayed for EM66. The bars above the peaks indicate the elution position of recombinant EM66, as indicated in A.

Figure 3

 3 Figure 3 Photomicrographs showing the distribution of EM66-like immunoreactivity in the rat pituitary. Consecutive sections of a rat pituitary gland were incubated with the EM66 antiserum (A) or with the same antiserum preabsorbed overnight with 10 Ϫ6 M purified EM66 (B). (C) Intense immunofluorescence labeling was observed in many cells of the anterior lobe (al) and moderate labeling of melanotrophs in the intermediate lobe (il) as well as nerve fibers in the neural lobe (nl). (D) Higher magnification showing the distribution of EM66-like immunoreactivity in the intermediate and neural lobes. Bars: A,B ϭ 150 m; C ϭ 50 m; D ϭ 10 m.

Figure 5

 5 Figure 5 Photomicrographs showing the distribution of EM66-and PRLlike immunoreactivity in the rat pituitary. Consecutive sections through the anterior lobe of the pituitary were immunostained with antisera against EM66 (A) and PRL (B). Several lactotrophs were immunoreactive to EM66 (arrowheads). Bars ϭ 20 m.

Figure 4

 4 Figure 4Confocal laser scanning microscope photomicrographs comparing the distribution of EM66 and LH␤ immunoreactivity in the rat pituitary. Sections through the anterior lobe were labeled with a rabbit antiserum against EM66 (A) revealed with DAR/TXR and with a monoclonal antibody against the LH␤ subunit (B) revealed with GAM/FITC. Combination of the two images acquired in A and B showed the presence of EM66-like immunoreactivity in all gonadotrophs (C). A few cells that were stained by the EM66 antibody were not immunoreactive to LH␤ (arrowheads). Bars ϭ 10 m.

Figure 7

 7 Figure 7 Confocal laser scanning microscope photomicrographs comparing the distribution of EM66 and ACTH-IR in the rat pituitary. Sections through the anterior lobe were labeled with a rabbit antiserum against EM66 (A) revealed with DAR/TXR and with a sheep antiserum against ACTH (B) revealed with DAS/FITC. Combination of the two images acquired in A and B showed the absence of EM66-like immunoreactivity in corticotrophs (C). Bars ϭ 40 m.

Figure 6

 6 Figure 6Immunofluorescence labeling of EM66 and TSH in the rat pituitary. Consecutive sections through the anterior lobe of the pituitary were immunostained with antisera against EM66 (A) and TSH␤ (B). A thyrotroph that was stained by the EM66 antibody is indicated by an arrowhead. Bars ϭ 20 m.

Figure 8

 8 Figure 8Immunoelectron microscopic localization of EM66 in the anterior pituitary gland. (A) Gold particles are located over the small and large secretory granules (arrowheads) of a gonadotroph (Gn). An adjacent somatotroph (GH) is unlabeled. (B) A few gold particles is also observed over the secretory granules of a prolactin cell (PRL). An adjacent gonadotroph (Gn) is more strongly labeled. Original magnification ϫ50,000.

Figure 9

 9 Figure 9 Photomicrographs showing the distribution of EM66-like immunoreactivity in the rat adrenal gland. (A) Intense immunofluorescence labeling was observed in chromaffin cells in the medulla (m). (B) Rays of EM66-IR chromaffin cells were also observed in the cortex (cx), whereas adrenocortical cells were devoid of labeling. Bars: A ϭ 100 m; B ϭ 50 m.

Figure 10

 10 Figure 10 Confocal laser scanning microscope photomicrographs comparing the distribution of EM66, PNMT, and TH immunoreactivity in the rat adrenal gland. Sections through the adrenal were labeled with rabbit antiserum against EM66 (A) or PNMT (D) revealed with DAR/ TXR and with a monoclonal antibody against TH (B,E) revealed with GAM/FITC. Combination of the two images acquired in A and B, and in D and E, revealed the presence of EM66 and PNMT in the same adrenochromaffin cells, i.e., adrenergic cells (arrowheads, C and F). Bars ϭ 100 m.

Figure 11

 11 Figure 11 Immunoelectron microscopic localization of EM66 in the adrenal gland. (A) Gold particles are located over secretory granules (arrowhead) of a chromaffin cell (Ch) whereas an adjacent steroid-secreting cell (S) is unlabeled. Original magnification ϫ25,000. (B) Higher magnification of a portion of the chromaffin cell. Original magnification ϫ50,000.

  Ait-Ali D, Turquier V, Alexandre D, Grumolato L,Jegou S, Vaudry H, Anouar Y (2002) Molecular characterization of frog chromogranin B reveals conservation of selective sequences encoding potential novel regulatory peptides. FEBS Lett 511:127-132 Anouar Y, de Monti M, Galindo E, Benie T, Aunis D, Duval J (1991) Regulatory effects of estrogens on the pituitary content of secretogranin II and chromogranin A. J Neuroendocrinol 3:232-235 Anouar Y, Desmoucelles C, Yon L, Leprince J, Breault L, Gallo-Payet N, Vaudry H (1998) Identification of a novel secretogranin II-derived peptide (SgII 187-252 ) in adult and fetal human adrenal glands using antibodies raised against the human recombinant peptide. J Clin Endocrinol Metab 83:2944-2951 Anouar Y, Duval J (1991) Differential expression of secretogranin II and chromogranin A genes in the female rat pituitary through sexual maturation and estrous cycle. Endocrinology 128:1374-1380 Anouar Y, Duval J (1992) Direct estradiol down-regulation of secretogranin II and chromogranin A mRNA levels in rat pituitary cells. Mol Cell Endocrinol 88:97-104 Anouar Y, Jegou S, Alexandre D, Lihrmann I, Conlon JM, Vaudry H (1996) Molecular cloning of frog secretogranin II reveals the occurrence of several highly conserved potential regulatory peptides. FEBS Lett 394:295-299 Berka JL, Kelly DJ, Robinson DB, Alcorn D, Marley PD, Fernley RT, Skinner SL (1996) Adrenaline cells of the rat adrenal cortex and medulla contain renin and prorenin. Mol Cell Endocrinol 119:175-184 Blazquez M, Bosma PT, Chang JP, Docherty K, Trudeau VL (1998) Gamma-aminobutyric acid up-regulates the expression of a novel secretogranin-II messenger ribonucleic acid in the goldfish pituitary. Endocrinology 139:4870-4880 Bornstein SR, Ehrhart-Bornstein M (1992) Ultrastructural evidence for a paracrine regulation of the rat adrenal cortex mediated by the local release of catecholamines from chromaffin cells. Endocrinology 131:3126-3128 Bornstein SR, Ehrhart-Bornstein M, Usadel H, Böckmann M, Scherbaum WA (1991) Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland. Cell Tissue Res 265:1-9 Bornstein SR, Tian H, Haidan A, Bottner A, Hiroi N, Eisenhofer G, McCann SM, et al. (2000) Deletion of tyrosine hydroxylase gene reveals functional interdependence of adrenocortical and chromaffin cell system in vivo. Proc Natl Acad Sci USA 97:14742-

Acknowledgments

Supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM U413) and the Conseil Régional de Haute-Normandie. We are very grateful to Dr JL Do Régo for CLSM data analysis and Ms H Lemonnier for skillful technical assistance. We thank Drs JF Roser (Davis, CA), and J Cote (Laval University, Quebec, Canada) for generously supplying the LH␤ antibodies and the ACTH antiserum, respectively.


Erwan Thouënnon

Alice Pierre

Laurent Yon

Youssef Anouar
email: youssef.anouar@univ-rouen.fr

Erwan Thou

Á A Pierre

Á L Yon

Expression of Trophic Peptides and Their Receptors in Chromaffin Cells and Pheochromocytoma

Keywords: Chromaffin cells, Pheochromocytomas, Trophic peptides, Tumorigenesis, Malignancy

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Introduction

Pheochromocytomas are neuroendocrine tumors arising from chromaffin cells of the adrenal medulla or extraadrenal locations. These tumors exhibit impaired control of peptide and hormone biosynthesis and secretion, resulting in an exacerbated production of these factors [START_REF] Thoue ¨nnon | Identification of potential gene markers and insights into the pathophysiology of pheochromocytoma malignancy[END_REF]. In particular, the main clinical symptoms indicating the occurrence of a pheochromocytoma are provoked by excessive release of catecholamines, dopamine, norepinephrine, and epinephrine, into the bloodstream. Along with catecholamines, pheochromocytomas produce and secrete numerous peptides. These peptides are also secreted along with catecholamines in physiological conditions, in order to participate in homeostatic regulations. Thus, peptides, such as neuropeptide Y (NPY) and adrenomedullin (AM), play an important role in the stress response [START_REF] Edvinsson | Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat[END_REF][START_REF] Shimosawa | Adrenomedullin and its related peptide[END_REF]. Other peptides, such as pituitary adenylate cyclaseactivating polypeptide (PACAP) can act as neurotransmitters in chromaffin cells [START_REF] Lamouche | PACAP release from the canine adrenal gland in vivo: its functional role in severe hypotension[END_REF][START_REF] Payet | PAC1 receptor activation by PACAP-38 mediates Ca 2? release from a cAMP-dependent pool in human fetal adrenal gland chromaffin cells[END_REF]. In addition, these peptides can also act locally as trophic factors. In particular, NPY, AM, and PACAP have been shown to be involved in hormone production and secretion, as well as proliferation, survival or differentiation of numerous cell types [START_REF] Shimosawa | Adrenomedullin and its related peptide[END_REF][START_REF] Dumont | An overview of neuropeptide Y: pharmacology to molecular biology and receptor localization[END_REF][START_REF] Vaudry | Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery[END_REF]. In pathophysiological conditions, alteration of NPY, AM, and PACAP expression, production and regulated secretion may lead to potential paracrine, autocrine or endocrine defects. Although these peptides have been extensively studied in different tumors, such as brain tumors, breast carcinomas, prostate cancer, and neuroblastomas, their roles in pheochromocytomas, where they are present in very high quantities, remain poorly understood accordance with the chemokines receptor nomenclature [START_REF] Bleul | The lymphocyte chemoattractant SDF-1 is a ligand for LESTR/fusin and blocks HIV-1 entry[END_REF].

In the human adrenal medulla, the receptors Y1, Y2, Y4, and Y5 are expressed and functional, indicating that NPY exerts autocrine effects in this tissue [START_REF] Cavadas | NPY regulates catecholamine secretion from human adrenal chromaffin cells[END_REF][START_REF] Korner | High expression of neuropeptide y receptors in tumors of the human adrenal gland and extraadrenal paraganglia[END_REF][START_REF] Spinazzi | Neuropeptide-Y and Y-receptors in the autocrine-paracrine regulation of adrenal gland under physiological and pathophysiological conditions (Review)[END_REF]. The expression of the CXCR4 in this tissue has not been shown.

Few studies have reported on the effect of NPY on chromaffin cells. In human and murine chromaffin cells in primary culture, NPY is able to stimulate catecholamine secretion [START_REF] Cavadas | NPY regulates catecholamine secretion from human adrenal chromaffin cells[END_REF][START_REF] Cavadas | Deletion of the neuropeptide Y (NPY) Y1 receptor gene reveals a regulatory role of NPY on catecholamine synthesis and secretion[END_REF]. It has also been shown that NPY treatment of rat or bovine chromaffin cells in primary culture is able to inhibit cholinergic agonist-induced catecholamine secretion [START_REF] Hexum | Stimulation of cholinergic receptor mediated secretion from the bovine adrenal medulla by neuropeptide Y[END_REF][START_REF] Shimoda | Antiserum against neuropeptide Y enhances the nicotine-mediated release of catecholamines from cultured rat adrenal chromaffin cells[END_REF]. Strikingly, the opposite effect has been observed when the adrenal gland was perfused [START_REF] Hexum | Stimulation of cholinergic receptor mediated secretion from the bovine adrenal medulla by neuropeptide Y[END_REF]. In addition to its role in the regulation of catecholamine secretion, NPY is also able to act upstream, at the level of catecholamine biosynthesis, as shown by tyrosine hydroxylase (TH) overexpression observed in rat adrenal medulla in which NPY has been injected [START_REF] Hong | Role of neuropeptide Y in the regulation of tyrosine hydroxylase gene expression in rat adrenal glands[END_REF]. Moreover, a simultaneous treatment by ATP and NPY enhanced TH serine 31-phosphorylation, which would stabilize this enzyme [START_REF] Luke | Tyrosine hydroxylase phosphorylation increases in response to ATP and neuropeptide Y co-stimulation of ERK2 phosphorylation[END_REF].

In human pheochromocytomas, the existence of NPY-producing cells and high NPY concentrations have been reported (deS [START_REF] Senanayake | Production, characterization, and expression of neuropeptide Y by human pheochromocytoma[END_REF]. These high intra-tumoral quantities of NPY can be explained by a strong expression of the gene, associated with an efficient maturation of the precursor [START_REF] O'hare | Expression and precursor processing of neuropeptide Y in human pheochromocytoma and neuroblastoma tumors[END_REF][START_REF] Thoue ¨nnon | Expression of trophic amidated peptides and their receptors in benign and malignant pheochromocytomas: high expression of adrenomedullin RDC1 receptor and implication in tumoral cell survival[END_REF]. In pheochromocytoma patients, plasma NPY concentration is often elevated, particularly when the tumor is malignant. However, the percentage of patients with elevated plasma NPY levels is similar for benign and malignant tumors [START_REF] Grouzmann | Neuropeptide Y and neuron-specific enolase levels in benign and malignant pheochromocytomas[END_REF]). In tumoral tissue, NPY expression has been shown to be lower in malignant compared to benign pheochromocytomas [START_REF] Thoue ¨nnon | Expression of trophic amidated peptides and their receptors in benign and malignant pheochromocytomas: high expression of adrenomedullin RDC1 receptor and implication in tumoral cell survival[END_REF], although the differences were not found statistically significant in another study who also compared the two tumor subtypes (deS [START_REF] Senanayake | Production, characterization, and expression of neuropeptide Y by human pheochromocytoma[END_REF][START_REF] Thoue ¨nnon | Expression of trophic amidated peptides and their receptors in benign and malignant pheochromocytomas: high expression of adrenomedullin RDC1 receptor and implication in tumoral cell survival[END_REF]. In a subtype of hereditary pheochromocytomas associated with mutation of the von Hippel-Lindau gene, it has been shown that the expression level of NPY was significantly lower than in other hereditary and sporadic pheochromocytomas [START_REF] Cleary | Neuropeptide Y expression in phaeochromocytomas: relative absence in tumours from patients with von Hippel-Lindau syndrome[END_REF]). Together, these observations suggest that NPY levels are high but variable in pheochromocytoma and that this peptide could be differentially produced by different tumor subtypes.

We showed the expression of all NPY receptors in almost all studied benign and malignant pheochromocytomas, with no differences between benign and malignant tumors. Despite the fact that this expression was very [START_REF] Mo | PACAP expression and distribution in human breast cancer and healthy tissue[END_REF][START_REF] Moretti | PACAP and type I PACAP receptors in human prostate cancer tissue[END_REF][START_REF] Nakamura | Adrenomedullin: a tumor progression factor via angiogenic control[END_REF][START_REF] Ruscica | Role of neuropeptide Y and its receptors in the progression of endocrine-related cancer[END_REF].

In this review, we focused on the expression of NPY, PACAP, and AM and their receptors in adrenal medulla, and benign and malignant pheochromocytomas. We also described their effects on chromaffin cells and pheochromocytes, and how they could possibly be involved in adrenal gland tumorigenesis and pheochromocytoma metastasis.

NPY

NPY is a 36-amino acid peptide originally isolated from porcine brain [START_REF] Tatemoto | Neuropeptide Y-a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide[END_REF]. This neuropeptide acts as a co-transmitter, a neuromodulator and a neurohormone, and plays an important role in numerous physiological processes such as food intake, hormone secretion or regulation of the immune system [START_REF] Zukowska | Neuropeptide Y: a new mediator linking sympathetic nerves, blood vessels and immune system?[END_REF]. NPY is also considered as a growth factor for several cell types such as neuronal cells or smooth muscle cells [START_REF] Hansel | Neuropeptide Y functions as a neuroproliferative factor[END_REF][START_REF] Pons | Mitogenic actions of neuropeptide Y in vascular smooth muscle cells: synergetic interactions with the beta-adrenergic system[END_REF]. NPY is an angiogenic factor able to stimulate the proliferation and migration of endothelial cells, the formation of capillary tubes and the revascularization of ischemic tissues [START_REF] Lee | Neuropeptide Y induces ischemic angiogenesis and restores function of ischemic skeletal muscles[END_REF]. Moreover, NPY has already been shown to be involved in cell proliferation, angiogenesis, invasion, and metastasization of endocrine-related tumors [START_REF] Ruscica | Role of neuropeptide Y and its receptors in the progression of endocrine-related cancer[END_REF].

In human and other mammalian species, high concentrations of NPY have been found in the brain and the sympathetic nerve system, including adrenal medulla [START_REF] Allen | Neuropeptide Y (NPY) in the adrenal gland[END_REF]). In the latter tissue, NPY concentrations are higher than those measured in the adrenal cortex in all studied species [START_REF] Allen | Neuropeptide Y (NPY) in the adrenal gland[END_REF][START_REF] De Quidt | Neuropeptide Y in the adrenal gland: characterization, distribution and drug effects[END_REF].

NPY is co-secreted with norepinephrine and exerts a strong vasoconstrictor effect on cardiovascular system vessels, making this peptide an actor of the stress response [START_REF] Edvinsson | Neuropeptide Y: cerebrovascular innervation and vasomotor effects in the cat[END_REF].

So far, six receptors, Y1, Y2, Y3, Y4, Y5, and y6, have been described as NPY receptors. In human, the y6 receptor is a pseudogene, coding for a non-functional truncated protein [START_REF] Rose | Molecular genetic analysis of a human neuropeptide Y receptor. The human homolog of the murine ''Y5'' receptor may be a pseudogene[END_REF]. Moreover, there is some confusion regarding the human Y3 receptor, frequently described as a putative and uncharacterized receptor [START_REF] Fetissov | Distribution of NPY receptors in the hypothalamus[END_REF][START_REF] Larhammar | Molecular evolution of NPY receptor subtypes[END_REF]. However, a recent update of the National Center for Biotechnology Information (NCBI) website clarified the situation: the Y3 receptor does not seem to bind human NPY but exhibits a strong affinity to stromal cell-derived factor 1 (SDF1) chemokine. The Y3 receptor was thus renamed CXC chemokine receptor 4 (CXCR4), in weak, we cannot exclude the possibility of an autocrine effect of NPY in these tumors, given the elevated amounts of the peptide produced by pheochromocytomas (Thoue ¨nnon et al. 2010).

Altogether, these data suggest that NPY could act in an autocrine manner on catecholamine production and secretion and therefore participates in pathophysiological mechanisms involved in these tumors. Interestingly, although NPY does not seem to be involved in the proliferation or apoptosis of PC12 cells, arguing against a role of this peptide in chromaffin cell transformation, it has nevertheless been shown that NPY could participate in tumoral growth by stimulating neoangiogenesis [START_REF] Kitlinska | Differential effects of neuropeptide Y on the growth and vascularization of neural crest-derived tumors[END_REF][START_REF] Kitlinska | Neuropeptide Y in neural crest-derived tumors: effect on growth and vascularization[END_REF]).

PACAP

PACAP is an ubiquitous neuropeptide of 27 or 38-amino acid involved in numerous physiological functions [START_REF] Vaudry | Pituitary adenylate cyclase-activating polypeptide and its receptors: 20 years after the discovery[END_REF]). In rat, several studies showed the expression of the PACAP gene and the occurrence of peptide immunoreactivity in fibers innerving the adrenal medulla [START_REF] Nielsen | Prenatal expression of pituitary adenylate cyclase activating polypeptide (PACAP) in autonomic and sensory ganglia and spinal cord of rat embryos[END_REF]. Other studies showed the presence of PACAP in chromaffin cells of several mammalian species and in human fetal chromaffin cells [START_REF] Breault | Occurrence and effect of PACAP in the human fetal adrenal gland[END_REF].

Numerous in vitro and in vivo studies showed that PACAP acts as a neurotransmitter in order to regulate catecholamine secretion by chromaffin cells in physiological and pathophysiological conditions [START_REF] Lamouche | PACAP release from the canine adrenal gland in vivo: its functional role in severe hypotension[END_REF][START_REF] Payet | PAC1 receptor activation by PACAP-38 mediates Ca 2? release from a cAMP-dependent pool in human fetal adrenal gland chromaffin cells[END_REF][START_REF] Isobe | Expression of mRNAs for PACAP and its receptor in human neuroblastomas and their relationship to catecholamine synthesis[END_REF]). These effects of PACAP on catecholamine secretion are associated with increased expression of TH, dopamine b-hydroxylase, and phenylethanolamine N-methyltransferase (PNMT) genes, concomitantly with increased activity of these enzymes [START_REF] Tonshoff | Pituitary adenylate cyclase activating polypeptide (PACAP) regulates expression of catecholamine biosynthetic enzyme genes in bovine adrenal chromaffin cells[END_REF][START_REF] Mckenzie | Caffeine stimulates Ca(2?) entry through store-operated channels to activate tyrosine hydroxylase in bovine chromaffin cells[END_REF]. However, immunolabeling of TH and PNMT enzymes is similar in PACAP knock-out and wildtype mice, suggesting that the peptide does not exert an important role in the maintenance of the catecholaminergic phenotype of chromaffin cells and in the development of the adrenal medulla [START_REF] Hamelink | Pituitary adenylate cyclase-activating polypeptide is a sympathoadrenal neurotransmitter involved in catecholamine regulation and glucohomeostasis[END_REF].

PACAP also stimulates the expression and secretion of several neuropeptides such as VIP, galanin, the brain natriuretic peptide or enkephalins [START_REF] Babinski | Pituitary adenylate-cyclase activating polypeptide (PACAP) evokes longlasting secretion and de novo biosynthesis of bovine adrenal medullary neuropeptides[END_REF][START_REF] Lee | Pituitary adenylate cyclase-activating polypeptide regulation of vasoactive intestinal polypeptide transcription requires Ca 2? influx and activation of the serine/threonine phosphatase calcineurin[END_REF]Guillemot et al. 2006a). Moreover, it has been shown that treatment of bovine chromaffin cells by PACAP leads to increased expression and secretion of secretogranin II and its derived peptides secretoneurin and EM66 (Guillemot et al. 2006b).

In the rat pheochromocytoma PC12 cell line, PACAP is able to stimulate catecholamine secretion, to inhibit cell proliferation and to induce differentiation toward a sympathetic phenotype through molecular pathways similar to those activated by nerve growth factor [START_REF] Grumolato | PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells[END_REF]. In these cells, PACAP also exerts protective effects against ceramide, b-amyloid or hydrogen peroxide-induced stress [START_REF] Ghzili | Role of PACAP in the physiology and pathology of the sympathoadrenal system[END_REF]). In addition, the neuropeptide also stimulates gene transcription of TH and PNMT enzymes, the vesicular monoamine transporter 1, the vesicular acetylcholine transporter and chromogranin A and B, but inhibits the expression of secretogranin II [START_REF] Grumolato | PACAP and NGF regulate common and distinct traits of the sympathoadrenal lineage: effects on electrical properties, gene markers and transcription factors in differentiating PC12 cells[END_REF].

In human intra-and extra-adrenal pheochromocytomas, a PACAP-like immunoreactivity has been found in all studied tumors [START_REF] Takahashi | Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in pheochromocytomas[END_REF][START_REF] Fahrenkrug | PreproPACAP-derived peptides occur in VIP-producing tumours and co-exist with VIP[END_REF]. In half of pheochromocytoma cases, this immunoreactivity was high as compared to cortical or adrenal medulla [START_REF] Takahashi | Pituitary adenylate cyclase activating polypeptide (PACAP)-like immunoreactivity in pheochromocytomas[END_REF]. [START_REF] Isobe | Expression of mRNA for PACAP and its receptors in intra-and extra-adrenal human pheochromocytomas and their relationship to catecholamine synthesis[END_REF] showed that PACAP was expressed in 24 out of 30 studied tumors. In intra-adrenal pheochromocytomas, this expression was strongly correlated to those of TH and PNMT genes and to intra-tumoral epinephrine concentrations, suggesting that PACAP may regulate the expression of the genes encoding catecholamine-synthesizing enzymes. In a set of 25 benign and malignant pheochromocytomas and paragangliomas, we also showed the wide expression of PACAP, which was detected in 24 of these tumors. However, we did not observe significantly different expression levels between benign and malignant pheochromocytomas [START_REF] Thoue ¨nnon | Expression of trophic amidated peptides and their receptors in benign and malignant pheochromocytomas: high expression of adrenomedullin RDC1 receptor and implication in tumoral cell survival[END_REF]. In this pheochromocytoma cohort, we also showed that the levels of PACAP and NPY were correlated and that PACAP was able to increase the expression of NPY in PC12 cells (Fig. 1; Thoue ¨nnon et al., unpublished data). This observation suggests that PACAP could exert direct trophic effects but could also exert indirect effects through stimulation of the expression of other trophic peptides.

PACAP exerts its effects by binding to VIP/PACAP receptors (VPAC1-R and VPAC2-R) and the PACAPpreferring receptor (PAC1-R). In chromaffin cells, PAC1-R is the predominant receptor but VPAC2-R is also present [START_REF] Harmar | Distribution of the VPAC2 receptor in peripheral tissues of the mouse[END_REF]). PAC1-R has also been detected in human fetal chromaffin cells [START_REF] Yon | Localization, characterization, and second messenger coupling of pituitary adenylate cyclase-activating polypeptide receptors in the fetal human adrenal gland during the second trimester of gestation[END_REF]. PAC1-R is involved in the effects of PACAP on peptide and catecholamine biosynthesis in the adrenal medulla [START_REF] Ghzili | Role of PACAP in the physiology and pathology of the sympathoadrenal system[END_REF].

We showed the expression of the PAC1 receptor in all studied pheochromocytomas whereas that of VPAC1-R and VPAC2-R mRNAs was only detected in half of them. Moreover, mean PAC1-R expression levels were about 20-fold higher than those of VPAC1-R and VPAC2-R. This high expression of PAC1-R is in line with the data reported in a previous study showing that PAC1-R mRNA is found in 83% of tumors among a set of 43 pheochromocytomas these cells are under hypoxia conditions [START_REF] Kobayashi | Pathophysiological function of adrenomedullin and proadrenomedullin N-terminal peptides in adrenal chromaffin cells[END_REF].

So far, three AM receptors, also exhibiting affinity for the CGRP, have been found: the adrenomedullin receptor (ADMR), the receptor dog cDNA 1 (RDC1), and the calcitonin receptor-like receptor (CRLR) linked to the receptor activity-modifying proteins 1, 2 or 3 (RAMP1-3). CRLR association with RAMP2 or 3 allows formation of a receptor displaying higher affinity for AM than for CGRP, while association with RAMP1 results in the opposite effect (Kobayashi et al. 2001b). In rat adrenal medulla, CRLR and ADMR receptors are exclusively detected in noradrenergic cells, while AM is mainly detected in adrenergic cells, suggesting a paracrine role for this peptide [START_REF] Renshaw | Adrenomedullin receptor is found exclusively in noradrenaline-secreting cells of the rat adrenal medulla[END_REF].

Treatment of rat or human chromaffin cells with AM provokes catecholamine secretion [START_REF] Mazzocchi | Distribution, functional role, and signaling mechanism of adrenomedullin receptors in the rat adrenal gland[END_REF]. However, this effect has not been observed in primary cultures of bovine chromaffin cells (Kobayashi et al. 2001b). In addition, in dog, injection of AM in the adrenal gland does not influence the secretion of catecholamines, even if a stimulation of the splanchnic nerve or an injection of acetylcholine is performed at the same time [START_REF] Masada | Effects of adrenomedullin and PAMP on adrenal catecholamine release in dogs[END_REF]. These results indicate that AM could have a role in the exocytosis of catecholamines from chromaffin cells in some species but not in others.

In human pheochromocytomas, we were able to detect AM expression in all of the 25 tumors analyzed, without differences between benign and malignant tumors. We observed that AM exerts anti-apoptotic effects on human pheochromocytomas in primary culture and on PC12 cells [START_REF] Thoue ¨nnon | Expression of trophic amidated peptides and their receptors in benign and malignant pheochromocytomas: high expression of adrenomedullin RDC1 receptor and implication in tumoral cell survival[END_REF]. Moreover, although no data is available for pheochromocytomas, numerous studies showed the overexpression of AM by several cell lines during hypoxia conditions. This overexpression is associated and paragangliomas [START_REF] Reubi | Vasoactive intestinal peptide/pituitary adenylate cyclase-activating peptide receptor subtypes in human tumors and their tissues of origin[END_REF]. Another study showed the expression of PAC1-R in only 63% of the pheochromocytomas and paragangliomas analyzed, but this lower rate could be explained by the use of a less sensitive technique [START_REF] Isobe | Expression of mRNA for PACAP and its receptors in intra-and extra-adrenal human pheochromocytomas and their relationship to catecholamine synthesis[END_REF]. These results indicate that PAC1-R is the main receptor potentially mediating autocrine/paracrine effects of PACAP in pheochromocytomas. Interestingly, PAC1-R is involved in the proliferation of several tumoral cells and is the receptor mediating the neuroprotective effects of PACAP [START_REF] Dejda | Inhibitory effect of PACAP on caspase activity in neuronal apoptosis: a better understanding towards therapeutic applications in neurodegenerative diseases[END_REF].

Altogether, these data suggest that PACAP, through its PAC1 receptor, could play an important role into the pathophysiology of pheochromocytomas. PACAP may exert trophic and anti-apoptotic effects on tumoral cells and, may increase the biosynthesis and secretion of catecholamines and other trophic peptides in these tumors.

AM

AM is a 52-amino acid peptide originally isolated from a human pheochromocytoma, which exhibits a high sequence homology with calcitonin gene-related peptide (CGRP) [START_REF] Kitamura | Adrenomedullin: a novel hypotensive peptide isolated from human pheochromocytoma[END_REF]. AM is also present at high concentrations in the adrenal medulla, is secreted in the bloodstream and exerts hypotensive effects, acting on vasodilatation and increasing diuresis and sodium secretion in urine [START_REF] Shimosawa | Adrenomedullin and its related peptide[END_REF]. AM also possesses a proliferative and anti-apoptotic actions on some cell types [START_REF] Malendowicz | Endogenous adrenomedullin system regulates the growth of rat adrenocortical cells cultured in vitro[END_REF][START_REF] Uzan | Adrenomedullin is anti-apoptotic in osteoblasts through CGRP1 receptors and MEK-ERK pathway[END_REF].

In primary cultures of bovine chromaffin cells, it has been shown that AM is stored in dense core vesicles and released along with catecholamines upon stimulation (Kobayashi et al. 2001a). Moreover, there is an increase in AM and concomitant catecholamine secretion when with a role of AM in angiogenesis, including tumoral angiogenesis [START_REF] Zudaire | Adrenomedullin and cancer[END_REF].

We also detected very low ADMR and CRLR mRNA levels in all tumors analyzed. Moreover, expression of RAMP2 and 3 was also very weak, exhibiting 2-to 12-fold lower levels than RAMP1 expression levels, which suggests that in pheochromocytomas CRLR/RAMP association is more likely forming a receptor with affinity towards CGRP. On the contrary, RDC1 expression in these tumors was very high, showing 20-to 30-fold higher levels than those of ADMR and CRLR, indicating that RDC1 is likely the receptor mediating AM effects in pheochromocytomas [START_REF] Thoue ¨nnon | Expression of trophic amidated peptides and their receptors in benign and malignant pheochromocytomas: high expression of adrenomedullin RDC1 receptor and implication in tumoral cell survival[END_REF]. Moreover, RDC1 is overexpressed in malignant pheochromocytomas, and down-regulation of its expression in PC12 cells reduced the number of cells (Thoue ¨nnon et al. 2010). Interestingly, RDC1 has already been shown to be overexpressed in several cancers and to be associated with the malignant behavior of tumors, i.e., invasiveness, survival, proliferation, and neoangiogenesis [START_REF] Miao | CXCR7 (RDC1) promotes breast and lung tumor growth in vivo and is expressed on tumor-associated vasculature[END_REF][START_REF] Wang | The role of CXCR7/RDC1 as a chemokine receptor for CXCL12/SDF-1 in prostate cancer[END_REF]). In addition, Autelitano et al. have shown that the effects of AM on vascular smooth muscle cells and cardiac myocytes may involve RDC1 [START_REF] Autelitano | Cardiac expression of genes encoding putative adrenomedullin/calcitonin gene-related peptide receptors[END_REF][START_REF] Autelitano | Co-expression of prepro-adrenomedullin with a putative adrenomedullin receptor gene in vascular smooth muscle[END_REF]. Although no study has shown an association between RDC1 and angiogenesis in pheochromocytomas, it is interesting to note that expression levels of this receptor and those of the vascular endothelial growth factor are strongly and significantly correlated in a set of 13 pheochromocytomas (Fig. 2; Thoue ¨nnon et al., unpublished data).

Altogether, these data strongly suggest that AM and its RDC1 receptor could participate in the tumorigenesis of chromaffin cells, supporting neoangiogenesis or allowing tumoral cell survival. Moreover, overexpression of RDC1 in malignant pheochromocytomas suggests a role for this receptor into tumor metastasis.

Conclusion

There is growing evidence that several peptides may play an important role in the pathophysiology of pheochromocytoma. Peptides such as NPY, PACAP, and AM may act on catecholamine release but also on cell survival and growth, representing thus with their receptors valuable targets in order to design new therapeutic tools for the management of these neuroendocrine tumors.

Fig. 1

 1 Fig. 1 Correlation between expression levels of PACAP and NPY in a set of 25 pheochromocytomas (a) and effect of treatment of PC12 cells with PACAP on NPY expression (b). a This study performed by real-time PCR shows that PACAP and NPY expression were

Fig. 2

 2 Fig.2Correlation between the expression levels of RDC1 and VEGF in a set of 13 pheochromocytomas. This study performed by real-time PCR showed that DC1 and VEGF expression were strongly and significantly correlated in human pheochromocytomas. ** P \ 0.01; r = 0.7637

Acknowledgments This work was financially supported by the Association pour la Recherche sur le Cancer, Ligue Nationale de Recherche Contre le Cancer, The COMETE Network (PHRC no. 06179).