Effect of hypercalcemia on parafollicular cells in the rat thyroid gland

Hypercalcemia was induced in rats by the administration of A.T.10. We then determined the levels of total and ionized calcium and calcitonin in the serum, as well as performed ultrastructural observations and histochemical investigations of the calcitonin and neuron-specific enolase immunoreactivities in the stimulated parafollicular cells. The main aim of the study was to apply histochemical procedures to determine the immunoreactions of calcitonin gene-related peptide (CGRP), somatostatin and secretory protein-I in stimulated parafollicular cells. Immunoreactions of CGRP and calcitonin decreased strikingly in A.T.10-treated animals, whereas no visible changes were noted in somatostatin immunoreactivity. In the case of secretory protein-I, an insignificant increase of its immunoreactivity was observed in the treated animals. The cytophysiological significance of these results is discussed.     

Immunocytochemical studies on thyroid parafollicular cells in postnatal development of the rat

Studies were performed on Wistar strain rats aged 1-720 days. Immunocytochemical reactions were used to detect calcitonin, somatostatin, calcitonin gene-related peptide (CGRP), cholecystokinin, serotonin, neuron-specific enolase (NSE), secretory protein-I, chromogranin and Ca-binding protein. In the parafollicular cells of the rat, the presence of calcitonin, somatostatin, CGRP, NSE and secretory protein-I could be demonstrated. The number of parafollicular cells increased with the age of animals, and the increase was particularly pronounced in the early postnatal period and after the first year of age. The number of somatostatin-immunoreactive cells decreased after birth and increased again after the first year of age. The number of calcitonin-immunoreactive cells increased in the early postnatal period independently of the increase in parafollicular cell number, forming frequently tumor-like outgrowths in 2-year-old animals. A small proportion of these outgrowths contained no calcitonin even if they did contain somatostatin, CGRP and NSE immunoreactivity. Evident changes in immunoreactivity in the first days after birth may reflect the sudden change in environment and may be associated with growth and differentiation. In any period of life, CGRP- and NSE-immunoreactive cells have constituted the most numerous groups and, therefore, the respective antigens seem to represent the most suitable markers of parafollicular cells in the rat.     

Immunocytochemical studies on parafollicular cells of various mammals

Using specific antisera, calcitonin, calcitonin gene-related peptide (CGRP), somatostatin as well as neuron-specific enolase, chromogranin, secretory peptide I and calbindin (vitamin D-dependent calcium-binding protein) were looked for in parafollicular cells of rats, Syrian hamsters, Mongolian gerbils, mice, guinea pigs, rabbits and pigs. Calcitonin and CGRP were most invariably present in various species. Somatostatin was absent in mice and Mongolian gerbils and present in variable amounts in the remaining species. Neuron-specific enolase could not be detected in rabbits, while in the pigs and the Mongolian gerbils it could be demonstrated only in some parafollicular cells. Calbindin was present exclusively in parafollicular cells of guinea pigs. Chromogranin and secretory protein-I were present only in some animal species.     

Electron-immunocytochemical localization of calcitonin and calcitonin-gene-related peptide in human c cells of the thyroid

A preembedding immunocytochemical technique enabled us to demonstrate normal human parafollicular (C) cells at the electron-microscopic level. The normal human C cells had numerous large secretory granules with a diameter of approximately 200 nm, well-developed rough endoplasmic reticulum and Golgi complex in their cytoplasm. Calcitonin immunoreactivity and calcitonin-gene-related peptide (CGRP) immunoreactivity were present only in the C cells whose secretory granules were heavily labeled. Both calcitonin and CGRP immunoreaction deposits were seen in the cytosol but not in the cisterna of endoplasmic reticulum, Golgi apparatus or mitochondrial matrix. The two peptides produced from a single calcitonin gene were stored in the secretory granules of the C cells.     

Light- and electron-microscopical localization of calcitonin, calcitonin gene-related peptide, somatostatin and C-terminal gastrin/cholecystokinin immunoreactivities in rat thyroid

Parafollicular C cells of the rat thyroid contain several immunoreactive peptides including calcitonin (CT), calcitonin gene-related peptide (CGRP), somatostatin and a C-terminal gastrin/CCK immunoreactive epitope as shown at the light- and electron-microscopical levels. Adult thyroid C cells are strongly immunoreactive to CT and most of the cells also react strongly with CGRP antisera and weakly with a gastrin/CCK antiserum. The latter antiserum may cross-react with CGRP. This cross-reactivity probably only occurs at very high concentrations of CGRP observed in adult thyroid C cells, but not in intrathyroidal CGRP-containing nerves, nor in early neonatal C cells. In neonatal rats, somatostatin immunoreactive C cells are numerous and most of these cells are also CT and CGRP immunoreactive. In contrast, only few C cells display somatostatin immunoreactivity in adult rat thyroids. Sequential staining experiments revealed that some thyroidal C cells simultaneously express all four types of immunoreactivity. At the electron microscopical level, all of these immunoreactivities were observed in secretory granules of C cells. Double- and triple-staining experiments, moreover, documented that some peptides are co-localized in the same granules.     

Alteration of parafollicular (C) cells activity in the experimental model of hypothyroidism in rats

Our previous study has shown the alteration of C cells activity in rats with experimental model of hyperthyroidism. The aim of the present study was the evaluation of parafollicular cells activity in rats with hypothyroidism evoked by propylthiouracil (PTU) given in drinking water over 21 days. Histological, ultrastructural and immunocytochemical studies using specific antibodies against calcitonin and CGRP were performed on thyroid glands taken from experimental and control groups of rats. Moreover, in all animals the calcitonin plasma levels were evaluated by radioimmunoassay. After chronic administration of PTU, thyroid image showed predominant microfollicular hyperplasia and attenuated density of parafollicular cells. The intensity of immunocytochemical reactions for CT and CGRP were weaker in the majority of C cells in comparison to the control rats, in which strong immunocytochemical reaction was observed. Examination in the electron microscope reveals the features of hypoactivity both in follicular and parafollicular cells, in which the quantity and electron density of secretory granules were smaller in comparison to the control group. These microscopic changes were accompanied by a significant decrease of calcitonin plasma concentration. Alteration of C cells activity in the experimental model of hypothyroidism, accompanied by microfollicular hypertrophy, may point to the mutual cooperation between parafollicular and follicular cells.     

Parafollicular cells of rabbit thryoid store both calcitonin and somatostatin and resemble gut D cells ultrastructurally.

Both calcitonin and somatostatin have been detected immunohistochemically in rabbit parafollicular cells; only calcitonin has been found in the same cells of the dog, guinea-pig and man. Large amounts of a peptide radioimmunochemically identical with synthetic somatostatin have been detected in extracts of rabbit thyroid. The ultrastructural and staining features of rabbit parafollicular cells differ from those of parafollicular cells in other species, while resembling in part those of somatostatin D cells scattered in the rabbit stomach.     

Parafollicular cells of rabbit thyroid store both calcitonin and somatostatin and resemble gut D cells ultrastructurally

Summary Both calcitonin and somatostatin have been detected immunohistochemically in rabbit parafollicular cells; only calcitonin has been found in the same cells of the dog, guinea-pig and man. Large amounts of a peptide radioimmunochemically identical with synthetic somatostatin have been detected in extracts of rabbit thyroid. The ultrastructural and staining features of rabbit parafollicular cells differ from those of parafollicular cells in other species, while resembling in part those of somatostatin D cells scattered in the rabbit stomach.Supported in part by grants from the Italian Ministero della Pubblica Istruzione and Consiglio Nazionale delle Ricerche     

Light- and electron-microscopical localization of calcitonin,calcitonin gene-related peptide,somatostatin and C-terminal gastrin/cholecystokinin immunoreactivities in rat thyroid

Summary Parafollicular C cells of the rat thyroid contain several immunoreactive peptides including calcitonin (CT), calcitonin gene-related peptide (CGRP), somatostatin and a C-terminal gastrin/CCK immunoreactive epitope as shown at the light-and electron-microscopical levels. Adult thyroid C cells are strongly immunoreactive to CT and most of the cells also react strongly with CGRP antisera and weakly with a gastrin/CCK antiserum. The latter antiserum may cross-react with CGRP. This cross-reactivity probably only occurs at very high concentrations of CGRP observed in adult thyroid C cells, but not in intrathyroidal CGRP-containing nerves, nor in early neonatal C cells. In neonatal rats, somatostatin immunoreactive C cells are numerous and most of these cells are also CT and CGRP immunoreactive. In contrast, only few C cells display somatostatin immunoreactivity in adult rat thyroids. Sequential staining experiments revealed that some thyroidal C cells simultaneously express all four types of immunoreactivity. At the electron microscopical level, all of these immunoreactivities were observed in secretory granules of C cells. Double- and triple-staining experiments, moreover, documented that some peptides are co-localized in the same granules.     

Effect of Follicular Cells on Calcitonin Gene Expression in Thyroid Parafollicular Cells in Cell Culture

Expression of calcitonin (CT) gene in thyroid parafollicular cells involves alternate formation of CT mRNA or CGRP mRNA. High amounts of CT mRNA are formed only in thyroid gland and formation of CGRP mRNA dominates in the remaining organs. Apart from paracrine and endocrine factors, mRNA formation on the CT gene seems to be affected also by direct contacts with other cells present in the thyroid gland, in which parafollicular cells are located next to follicular cells.The present study aimed at examining whether thyroid follicular cells affect formation of mRNAs for CT and CGRP in parafollicular cells. The studies were performed in cell cultures. A parafollicular cell line (TT cells) and a follicular cell line (F6BTY cells) served as the experimental model. For comparison, co-cultures with fibroblasts, 3T3 cells, and malignant melanoma, MM cells, were also examined. CT gene expression was examined at the level of mRNAs (in situ hybridization and morphometric studies) and at the level of hormones (immunocytochemistry, morphometric studies and radioimmunological estimation of hormone levels in the medium).The immunocytochemical and hybridocytochemical studies, in line with morphometry studies, demonstrated that F6BTY and 3T3 cells were capable of affecting mRNA production for CT and CGRP and that they changed the ratio of CGRP/CT secretion by TT cells, as a sequel of contact between the two cell types and due to mediation of secreted substances. On the other hand, the malignant melanoma MM cells showed no effect on the secretion ratio.Our study seems to indicate that control of mRNA formation from CT gene may involve not only humoral factors but also direct contacts with other cells, which may explain differences in expression of the gene between cells localized in different organs.     

Single cellular origin of somatostatin and calcitonin in the rat thyroid gland

Summary Immunostaining of thin serial paraffin sections has shown that somatostatin is present in the same parafollicular cells as calcitonin in the adult rat thyroid gland. The number of cells containing both peptides is much smaller than the number containing calcitonin but not somatostatin.     

Inhibition of mitogen-stimulated T lymphocyte proliferation by calcitonin gene-related peptide

The effect of calcitonin gene-related peptide (CGRP) on mouse lymphocyte proliferation stimulated by mitogens was studied. CGRP (10(-10)-10(-7) M) dose-dependently inhibited the proliferative response of mouse lymph node cells and spleen cells stimulated by T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA), whereas a B cell mitogen lipopolysaccharide (LPS) did not inhibit this response. The maximal inhibition by this peptide was 50% to 80% at 10(-8) and 10(-7) M. The addition of 10(-8) and 10(-7) M CGRP to lymph node cell cultures 24 hr after stimulation with Con A or PHA also had a significant inhibitory effect on the proliferative response. Furthermore, in the same concentration range (10(-10)-10(-7) M) CGRP increased intracellular cyclic AMP concentration in nylon wool nonadherent cells, but not in nylon wool adherent cells. CGRP had no significant effect on intracellular cyclic GMP concentration. In addition, specific binding of CGRP was observed in mouse spleen cells. Our present study suggests that CGRP inhibits the proliferative response of T lymphocytes to the mitogens by interacting with cell receptors coupled with adenylate cyclase. CGRP may be implicated in the regulation of T cell function.     

Localization of calcitonin gene-related peptide and islet amyloid polypeptide in the rat and mouse pancreas

Summary It was previously demonstrated that the two chemically related peptides calcitonin gene-related peptide (CGRP) and islet amyloid polypeptide (IAPP) both occur in the pancreas. We have now examined the cellular localization of CGRP and IAPP in the rat and the mouse pancreas. We found, in both the rat and the mouse pancreas, CGRP-immunoreactive nerve fibers throughout the parenchyma, including the islets, with particular association with blood vessels. CGRP-immunoreactive nerve fibers were regularly seen within the islets. In contrast, no IAPP-immunoreactive nerve fibers were demonstrated in this location. Furthermore, in rat islets, CGRP immunoreactivity was demonstrated in peripherally located cells, constituting a major subpopulation of the somatostatin cells. Such cells were lacking in the mouse islets. IAPP-like immunoreactivity was demonstrated in rat and mouse islet insulin cells, and, in the rat, also in a few non-insulin cells in the islet periphery. These cells seemed to be identical with somatostatin/CGRP-immunoreactive elements. In summary, the study shows (1) that CGRP, but not IAPP, is a pancreati neuropeptide both in the mouse and the rat; (2) that a subpopulation of rat somatostatin cells contain CGRP; (3) that mouse islet endocrine cells do not contain CGRP; (4) that insulin cells in both the rat and the mouse contain IAPP; and (5) that in the rat, a non-insulin cell population apparently composed of somatostatin cells stores immunoreactive IAPP. We conclude that CGRP is a pancreatic neuropeptide and IAPP is an islet endocrine peptide in both the rat and the mouse, whereas CGRP is an islet endocrine peptide in the rat.     

Co-occurrence of immunoreactive calcitonin and calcitonin gene-related peptide in neuroendocrine cells of rat lungs

Summary Neuroendocrine cells of the lung, occurring singly or in clusters known as neuroepithelial bodies, contain a variety of biologically active compounds, including several neuropeptides. We have investigated the localization of calcitonin and calcitonin gene-related peptide (CGRP) within single and grouped neuroendocrine cells in the respiratory epithelium of rats by an immunohistochemical double-staining technique which uses specific antisera raised in heterogeneous animal species. Calcitonin- and CGRP-immunoreactivities were nearly totally co-localized in both single neuroendocrine cells and neuroepithelial bodies. CGRP-immunoreactivity was also present in neurons in the jugular, nodose and dorsal root ganglia. The calcitonin-immunoreactivity in neuroendocrine cells, as in thyroid parafollicular (C) cells, was abolished by preincubation of the anticalcitonin serum with synthetic calcitonin. The CGRP-immunoreactivity in neuroendocrine cells and in the neuronal cells was abolished by preincubation of anti-CGRP serum with synthetic CGRP. Thus, while the calcitonin gene is expressed exclusively or predominantly as either calcitonin or CGRP in all other tissues except thyroid C-cells, our results strongly suggest that both peptides are expressed in the rat bronchopulmonary neuroendocrine cells.     

Immunocytochemical localization of prohormone convertases PC1 and PC2 in the mouse thyroid gland and respiratory tract.

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.     

Ultrastructural localization of calcitonin, somatostatin and serotonin in parafollicular cells of rat thyroid

Summary Three hormones were demonstrated in ultrathin sections of the rat thyroid using immunocytochemical methods with either a PAP complex or a protein A-gold complex as the tabel. In control rats, calcitonin was found to be present in all parafollicular cells and somatostatin in occasional cells. In rats pretreated with 5-hydroxytryptophan, serotonin was detected in all parafollicular cells as well. In serial ultrathin sections, the three hormones were seen to be localized in the same secretory granules.     

Immunohistochemical study of a large molecular fragment of thyroglobulin in parafollicular cells

Thyroglobulin-like immunoreactivity of the parafollicular cells was studied by an immunoperoxidase bridge technique using antisera against dog thyroglobulin fragments. 1. The dog parafollicular cells were specifically stained by anti-peak I (27S and larger components fraction) antiserum absorbed with peak II (19S fraction). By this method, they were easily distinguishable from the non-reactive follicular cells and colloid droplets. More sensitive staining of the parafollicular cells was possible with anti-peak I' (larger components fraction) antiserum. The staining reactions indicated that the antigenic material responsible for immunoreactivity of the parafollicular cells was due to larger molecular components of thyroglobulin corresponding to 32S, 37S or greater than 37S, and was not due to either the 19S thyroglobulin or to the 27S iodoprotein. 2. A conspicuous decrease of the immunoreactive material in the parafollicular cells occurred in the dog after both chronically induced hypercalcemia and antithyroid drug treatment. This coincided with movement of secretory granules containing calcitonin as shown by staining with silver impregnation, HCl-basic dye, and lead-hematoxylin. 3. The antisera against larger molecular components of dog thyroglobulin showed a high degree of cross-reactivity to the parafollicular cells of most of the mammalian species investigated; rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human.     

Calcitonin and calcitonin gene-related peptide immunoreactivity and colocalization in newborn cat lung.

Calcitonin (CT) and calcitonin gene-related peptide (CGRP) are generated by alternate RNA processing from alpha and beta CT/CGRP genes. In this report, an immunocytochemical investigation was undertaken on the occurrence and distribution of immunoreactive CT as well as its colocalization with CGRP in newborn cat bronchopulmonary endocrine cells. A widespread distribution of solitary endocrine cells and neuroepithelial bodies immunostained for CT was recorded within the lung. In all animals studied, CT immunoreactivity represents a subpopulation of CGRP positive cells, while the intrapulmonary nerve fibers contain only CGRP. To the best of our knowledge, this is the first time that CT and its colocalization with CGRP have been demonstrated immunocytochemically in the cat lung. Our results indicate, that different molecular processing of both CT/CGRP genes may be represented by different patterns in the cellular immunoreactivity of the synthetized peptides.