ARTICLE |
Correspondence to: Shigeyasu Tanaka, Dept. of Biology, Faculty of Science, Shizuoka University, Ohya 836, Shizuoka 422-8529, Japan. E-mail: sbstana@ipc.shizuoka.ac.jp
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Summary |
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We examined immunocytochemical localization of the prohormone convertases, PC1 and PC2, in the thyroid gland and respiratory tract of the adult mouse using the indirect enzyme- and immunogold-labeled antibody methods for light and electron microscopy, respectively. In the thyroid gland, PC1- and/or PC2-immunoreactive cells were cuboidal, scattered in the follicular epithelium and in the interfollicular spaces. When serial sections were immunostained with anti-calcitonin, anti-PC1, anti-calcitonin-gene-related-peptide (CGRP), and anti-PC2 sera, respectively, localization of both PC1 and PC2 was restricted to the calcitonin/CGRP-producing parafollicular cells. In the respiratory tract, only PC1 immunoreactivity was observed in the basal granulated neuroendocrine cells, which were scattered in the tracheal epithelium. Consecutive sections immunostained with anti-PC1 and anti-CGRP sera showed that a subpopulation of these PC1-immunoreactive cells contained CGRP. Double immunogold electron microscopy of the thyroid parafollicular cells revealed that calcitonin- and/or CGRP-immunopositive secretory granules were also labeled with both PC1 and PC2. These findings suggest that procalcitonin is proteolytically cleaved by PC2 alone or by PC2 together with PC1, and that the proCGRP is cleaved by PC1.
(J Histochem Cytochem 50:903909, 2002)
Key Words: PC1, PC2, parafollicular cell (C-cell), thyroid gland, basal granulated cell, respiratory tract, immunocytochemistry, mouse
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Introduction |
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THE PROHORMONE CONVERTASES PC1 (also called PC3) and PC2 are responsible for the proteolytic cleavage of a large precursor molecule at paired basic sites to yield bioactive peptides in endocrine cells and neurons (
Calcitonin is a peptide hormone that regulates the balance of serum calcium, which is secreted primarily from parafollicular cells (C-cells) in the thyroid gland (
It has not yet been fully confirmed whether or not PC1 and/or PC2 are involved in the limited proteolysis of the precursors of calcitonin and CGRP. As far as we know, there has been only one IHC study, showing the presence of PC1 and PC2 in the normal human C-cells and medullary thyroid carcinomas (
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Materials and Methods |
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Animals
Normal adult male ICR mice, weighing about 40 g at 11 weeks of age, were housed under controlled environmental conditions (22C; L:D 12 hr:12 hr) and were allowed food and water ad libitum. Under sodium pentobarbital anesthesia, the trachea, with attached thyroid glands and parathyroid glands, was quickly removed from five mice and processed for light and electron microscopic ICC. All animal experiments were in compliance with the NIH Guide for the Care and Use of Laboratory Animals.
Antibodies
The antibodies used in this study were prepared in rabbits and characterized as described previously: anti-PC1 serum (ST-28) and anti-PC2 serum (ST-29) against synthetic peptides covering amino acids 442459 and amino acids 613629 of the respective mouse proteins (
Light Microscopic ICC
The organs were fixed by immersion in BouinHollande sublimate for 2 days. After dehydration and embedding in Paraplast, serial 5-µm-thick transverse sections of the trachea were cut, including both lobes of the thyroid gland and parathyroid glands, and mounted on gelatin-coated slides. The deparaffinized sections were treated with iodine alcohol and sodium pyrosulfite to eliminate the sublimate and then incubated in a solution of 0.3% H2O2 in methanol for 30 min to inhibit endogenous peroxidase activity. After rinsing with distilled water (DW) followed by PBS (0.01 M sodium phosphate buffer and 0.14 M NaCl, pH 7.5), the sections were immunostained by the streptavidinbiotin method. To amplify the signals for PC1 and PC2, the sections were treated with Gomori's oxidation mixture according to the procedure of
Adjacent serial sections were immunostained to identify parafollicular cells using the same method with rabbit anti-salmon calcitonin antiserum (1:10,000) and anti-CGRP serum (1:2000), respectively.
The specificity of the anti-PC1 and anti-PC2 sera was also tested using an ICC preabsorption test. Diluted antisera were preabsorbed with their corresponding peptides at a final concentration of 0.011 mg/ml at 4C for 16 hr before ICC studies.
Electron Microscopic ICC
One mm3 pieces of the thyroid glands were fixed with a mixture of 0.5% glutaraldehyde and 4% paraformaldehyde in 0.05 M cacodylate buffer, pH 7.4, for 2 hr at 4C and then dehydrated with a graded ethanol series and embedded in LR White (London Resin; Basingstoke, UK). Pale gold ultrathin sections were cut with an ultramicrotome (LKB 2088 Ultrotome V) and mounted on gold grids. Immunolabeling was performed by the two-face double-labeling method (
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Results |
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PC1- or PC2-immunopositive cells were observed in the thyroid glands (Fig 1) and respiratory tract (Fig 2). In the absorption test, the immunopositive materials obtained with both antisera were completely eliminated when 0.01 mg/ml of the respective antigen peptide was used as an adsorbent (data not shown).
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In the thyroid glands, PC1- or PC2-positive cells were round, scattered in the follicular epithelium and interfollicular space. PC1 immunoreactivity varied from cell to cell, suggesting a difference in PC1 content among the cells (Fig 1b). PC2 immunoreactivity was very faint (Fig 1d). When four serial sections of thyroid glands were immunostained with anti-calcitonin (Fig 1a), anti-PC1 (Fig 1b), anti-CGRP (Fig 1c), and anti-PC2 (Fig 1d), both PC1 and PC2 were localized in most of the C-cells containing both calcitonin and CGRP. However, the CGRP immunoreactivity was moderate or faint, whereas the calcitonin immunoreactivity was always strong.
In the respiratory tract, many PC1-positive cells were scattered in the tracheal epithelium (Fig 2a) and in the epithelium covering the ducts of the tracheal glands (Fig 2b), and a few CGRP-positive cells were seen located in the tracheal epithelium, but no PC2- or calcitonin-positive cells were observed (Fig 2d). When consecutive sections were immunostained with anti-PC1 and anti-CGRP, respectively, only a small population of PC1-positive cells corresponded with the CGRP-positive cells (Fig 2a and Fig 2c).
Although several segments of CGRP-immunopositive nerve terminals were seen around thyroid follicles and in the subepithelial connective tissue of the trachea, they were not stained with either PC1 or PC2 antiserum (data not shown). On the other hand, several histological studies have shown that C-cells are localized in the parathyroid gland of some mammals (
The electron microscopic observations were performed to extend the light microscopic results. Immunoelectron microscopy of the thyroid C-cells showed many electron-dense secretory granules packed in the cytoplasm (Fig 3a). In double labelings, both PC1 and PC2 were co-localized in the same granules, although the PC2-labels had a remarkably low density (Fig 3c). The double labelings for PC1 and calcitonin or for PC1 and CGRP showed that PC1 coexisted with calcitonin and CGRP in most of the secretory granules (Fig 3a, Fig 3b, and Fig 3d), but fewer secretory granules were labeled for CGRP than for calcitonin (Fig 3d). No or few labels were seen in other organelles.
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Discussion |
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The present study, using specific polyclonal antisera raised against PC1 and PC2, clearly demonstrates that C-cells in the thyroid gland and basal granulated cells in the respiratory tract express these convertases. Both antibodies against calcitonin and CGRP marked almost all of the C-cells in the mouse thyroid, even though CGRP immunoreactivity is not always detected in all thyroid C-cells in several animals, including the mouse (
It is well known that the granin family members chromogranin A (CgA) and secretogranin II (SgII) play various important roles in the regulatory mechanism of secretion in many endocrine cells, although their exact function has not yet been elucidated (
It is known that somatostatin is also produced in thyroid C-cells (
There are several reports showing that neuroendocrine cells in the tracheal epithelium and lung have immunoreactivity for bombesin, calcitonin, somatostatin, and Leu-enkephalin, as well as for CGRP (
In the present study we were unable to stain the CGRP-immunopositive nerve fibers by either PC1 or PC2 antiserum; the reason for this is unclear.
In the present immunoelectron microscopic study, a small population of secretory granules in the C-cells were labeled with the antiserum against CGRP, unlike the heavy labeling for calcitonin, although most of the C-cells displayed similar immunoreactivity for both the hormones by light microscopy. This low labeling density for CGRP was also consistent with a previous report in the bat thyroid C-cells (
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Acknowledgments |
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Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (SK and ST).
Received for publication September 26, 2001; accepted February 9, 2002.
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