ARTICLE |
Correspondence to: Hubert Vaudry, European Institute for Peptide Research (IFRMP23), Lab. of Cellular and Molecular Neuroendocrinology, INSERM U413, UA CNRS, University of Rouen, 76821 Mont-Saint-Aignan, France. E-mail: hubert.vaudry@univ-rouen.fr
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Summary |
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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. (J Histochem Cytochem 51:10831095, 2003)
Key Words: secretogranin II, chromogranins, novel peptides, EM66, pituitary cells, adrenal medulla, immunohistochemistry, electron microscopy, HPLC analysis
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Introduction |
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SECRETOGRANIN II (SgII) belongs to the chromogranin/secretogranin protein family whose members occur in most endocrine, neuroendocrine, and nerve cells (
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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 (
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Materials and Methods |
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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 -80C.
Production of Recombinant EM66
A fusion protein containing the human EM66 peptide was produced in E. coli as previously described (
Antibodies
Antibodies against EM66 (code 736-1806) were raised in rabbits against the recombinant fusion protein MBPEM66, and their specificity has been established by Western blotting and IHC (-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-µm sections using a cryomicrotome (Frigocut; ReicherJung, 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 LHß antibody diluted 1:300, the ACTH antiserum diluted 1:500, the PRL antiserum diluted 1:2000, the TSHß 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 (10-6 M).
Immunoelectron Microscopy
Three-month-old adult male SpragueDawley rats (225250 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 (1 mm3). The tissues were rinsed overnight in the same buffer and postfixed for 1 hr in 0.2% OsO4. 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 (
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 x 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 C18 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 x g for 10 min at 4C. The supernatants were injected onto a 0.45 x 25-cm Vydac C18 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 µg 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 C18 cartridges using a gradient of acetonitrile (0100%) 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 µl goat anti-rabbit -globulins (1:30), 200 µl normal rabbit serum (1:150), and 500 µl of 5% polyethyleneglycol 8000. After a 2-hr incubation at RT, the mixture was centrifuged (5000 x g, 4C, 30 min), and the pellet containing the bound fraction was counted on a
-counter (LKB; Wallack, Rockville, MD).
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Results |
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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 (Fig 2). Recombinant EM66 eluted as a single peak with a retention time of 17.5 min (Fig 2A). A major EM66-IR peak exhibiting the same retention time as the recombinant peptide was detected in the adenohypophysial (Fig 2B) and adrenal (Fig 2C) gland extracts. A more hydrophobic EM66-immunoreactive compound eluting at 22.5 min was also resolved in the adenohypophysial gland extract (Fig 2B).
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Distribution of EM66-IR in the Pituitary
The EM66 antiserum strongly labeled many cells in the anterior lobe (Fig 3A and Fig 3C) and moderately stained most melanotrophs in the intermediate lobe of the rat pituitary (Fig 3C and Fig 3D). At a higher magnification, EM66-IR was also observed in nerve endings in the neural lobe (Fig 3D). No staining was seen when the EM66 antiserum was preabsorbed with 10-6 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 (Fig 3B).
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Co-incubation of pituitary sections with the anti-EM66 antiserum and the anti-LHß antibody revealed that virtually all gonadotrophs contain EM66-IR (Fig 4A4C). However, some EM66-IR cells did not exhibit any LHß-IR (Fig 4A4C), indicating that the peptide occurs in cells other than gonadotrophs. Incubation of consecutive sections of the adenohypophysis with the anti-EM66 (Fig 5A) and anti-PRL (Fig 5B) antisera showed that some EM66-IR cells were lactotrophs (Fig 5A and Fig 5B). Similarly, incubation of consecutive sections with the anti-EM66 (Fig 6A) and the anti-TSHß (Fig 6B) antibodies revealed that EM66-IR is present in thyrotrophs (Fig 6A and Fig 6B). On the contrary, corticotrophs that were immunostained with the ACTH antibodies (Fig 7B) were totally devoid of EM66-IR (Fig 7A7C).
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The different types of pituitary cells can readily be identified on the basis of their ultrastructural characteristics (
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Distribution of EM66-IR in the Adrenal Gland
The EM66 antibody strongly labeled the whole medulla (Fig 9A) and groups of cells located in the cortex (Fig 9A and Fig 9B) of the rat adrenal gland. Preabsorption of the primary antiserum with 10-6 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 (Fig 10A10C), indicating that EM66 is present in chromaffin cells. However, a few TH-positive cells were devoid of EM66-IR (Fig 10C). Double immunolabeling of adrenal slices with the anti-PNMT and anti-TH antibodies (Fig 10D10F) revealed that the EM66-negative chromaffin cells were also PNMT-negative (Fig 10C and Fig 10F), indicating that EM66 is present only in adrenergic cells and not in noradrenergic cells of the rat adrenal gland.
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Immunocytochemical labeling of chromaffin cells showed that EM66-IR was restricted to secretory granules (Fig 11A and Fig 11B). In contrast, adrenocortical cells did not exhibit any staining (Fig 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).
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Discussion |
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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 (
In the anterior pituitary, intense EM66-IR was observed in all gonadotrophs as revealed by double labeling using a monoclonal antibody against LHß. 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 (
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 (
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 (
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 (
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.
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Acknowledgments |
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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 LHß antibodies and the ACTH antiserum, respectively.
Received for publication October 1, 2002; accepted February 26, 2003.
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