Monoamines in rat thyroid parafollicular cells and the effect of vitamin D2-induced degranulation

Summary Thyroid parafollicular cells of normocalcemic and vitamin D₂-treated rats were investigated by electron microscopy and with the histochemical fluorescence technique of Hillarp and Falck.Administration of high doses of vitamin D₂ caused hypercalcemia and an extensive degranulation of the parafollicular cells.The formation and storage of monoamines in granulated and degranulated parafollicular cells was investigated by fluorescence microscopy after injection of monoamine precursors (DOPA, 5-HTP), alone or in combination with Ro 4-4602, nialamide or reserpine.No fluorescence was observed in parafollicular cells of untreated rats. l-DOPA and l-5-HTP (but not the corresponding D-amino acids) were taken up by a process closely linked to the decarboxylation of the amino acids to the corresponding amines (dopamine and 5-hydroxytryptamine). Treatment with vitamin D₂ did not seem to affect the formation of amines in the parafollicular cells or the formation and storage of amines in other cell systems investigated. The amine itself (dopamine) was not taken up by the parafollicular cells.In normocalcemic rats, the amine formed was retained in the cytoplasm of the parafollicular cells by a partially reserpine-resistant mechanism. The storage of amines is concluded to occur in association with the calcitonin-containing granules.In parafollicular cells of vitamin D₂-treated rats, a certain amount of amine was bound in the cytoplasm in the absence of typical granules. As a considerable amount of calcitonin is known to remain in the thyroid of vitamin D₂-treated rats, the present observations may indicate an association between the amine and the polypeptide hormone calcitonin, whether the latter is confined to typical granules or not.The present study was supported by grants B72-12X-3352-02 and B72-14X-2207-06B from the Swedish Medical Research Council and by grants from Magnus Bergwall's Foundation, Gustav and Majen Lundgren's Foundation, Wilhelm and Martina Lundgren's Foundation and from the Faculty of Medicine, University of Göteborg, Sweden. For skilful technical assistance we are indebted to Mrs. Kirsten Collin and Mr. Pär-Anders Larsson.     

SUBCELLULAR STORAGE ORGANELLES FOR 5-HYDROXYTRYPTAMINE IN PARAFOLLICULAR CELLS OF THE THYROID GLAND : The Effect of Drugs which Deplete the Amine

A study was made of the effect of the administration of reserpine and parachlorophenylalanine, an inhibitor of 5-hydroxytryptamine (serotonin; 5-HT), on the capacity of thyroid parafollicular cells to synthesize and store 5-HT. The two drugs were given to nonhibernating bats in doses which produced an equivalent degree of depletion of 5-HT from the thyroid. Tritiated 5-hydroxytryptophan, the precursor of 5-HT, was then given intravenously to assess the ability of parafollicular cell granules to take up and retain newly synthesized 5-HT. Reserpine, but not parachlorophenylalanine, decreased the amount of labeled 5-HT found in the thyroid and prevented autoradiographic labeling of parafollicular cell granules. Quantitative ultrastructural and stereological analysis demonstrated that the granules in untreated animals appeared to be nearly spherical prolate ellipsoids, with a uniformly electron-opaque inner matrix. In animals given reserpine, the axial ratio of the ellipsoidal granules increased greatly and a faint internal striation parallel to the long axis of the granules became apparent. Similar changes were not induced by parachlorophenylalanine. No other morphological changes in the thyroid epithelium were detected after administration of reserpine. This study confirms the association of 5-HT with the mature small secretory granules of thyroid parafollicular cells.     

Origin of cholinesterase-containing follicle cells and parafollicular cells of the developing thyroid gland in the rat

Summary The location of cholinesterase-containing cells in the thyroid gland and its precursors (median thyroid primordium and ultimobranchial bodies) has been investigated light-microscopically in rat embryos from the 13 to the 20th day of gestation.From the 13th to the 16th day of gestation the median thyroid primordium and the ultimobranchial bodies are distinct from each other. Cholinesterase-containing cells are found in both. On the 17th–18th day of gestation the reacting ultimobranchial cells spread into the median thyroid primordium where they take up a parafollicular position. At the 19th–20th day of gestation the distribution of cholinesterase-containing cells is as in the adult rat. The results seem to show that cholinesterase-containing follicular cells derive from the median thyroid primordium and cholinesterase-containing parafollicular cells from the ultimobranchial body.     

Thyrotropin-releasing hormone gene expression in normal thyroid parafollicular cells

The rat TRH gene encodes a 255-amino-acid precursor polypeptide, preproTRH, containing five copies of TRH and seven non-TRH peptides. Expression of this gene is well documented in the central nervous system, particularly in the hypothalamus. Thyroids also contain TRH immunoreactivity, but it is unknown whether this immunoreactivity results from expression of the TRH gene or from other genes encoding TRH-like products. Since the CA77 neoplastic parafollicular cell line expresses the TRH gene, we investigated whether TRH gene expression also occurs in normal thyroid parafollicular cells. Northern analysis of total thyroid RNA with a preproTRH-specific RNA probe identified a single hybridizing band the same size as authentic TRH mRNA found in hypothalamus and CA77 cells. Gel filtration analysis of thyroid extracts identified the same 7-kilodalton and 3-kilodalton species of immunoreactive preproTRH53-74 previously identified in hypothalamus and CA77 cells. Immunoreactive preproTRH115-151, not previously identified, was found in all three tissues. Part of this immunoreactivity comigrated with the synthetic preproTRH115-151 standard on gel filtration and reversed-phase HPLC. PreproTRH53-74 was localized to thyroid parafollicular cells by immunostaining. These findings demonstrate authentic TRH gene expression by normal rat thyroid parafollicular cells and establish the CA77 cell line as the only model system of a normal TRH-producing tissue. In addition to expanding the range of neuroendocrine peptides known to be produced by parafollicular cells, these results also suggest a potential paracrine regulatory role for TRH gene products within the thyroid.     

Fine structure of the fetal rat thyroid and parathyroid glands at term and during prolonged gestation

Summary The fine structure of the fetal rat thyroid and parathyroid glands was studied at term and during prolonged gestation, which was induced by subcutaneous injections of progesterone to the mothers from gestational days 20 through 24. At term, the follicular and parafollicular cells of the thyroid as well as cells of the parathyroid exhibited well developed cytoplasmic organelles. Morphological changes were not detected in either of the endocrine glands during prolonged gestation. The results are discussed in relationship to 1) thyroid follicular cell activity during stress and 2) the function of thyroid parafollicular and parathyroid cells in calcium homeostasis.Supported by the Medical Research Council of Canada Grant No. MA4740.     

Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane

Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino- N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH- treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP- driven H+ translocation into parafollicular granules isolated from TSH- stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.     

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     

Uptake of DOPA and 5-HTP in pancreatic islets and parafollicular cells studied by combined autoradiographic and fluorescence microscopic techniques

Summary White albino mice were simultaneously injected with ³H-5-HTP and unlabelled DOPA or ³H-DOPA and unlabelled 5-HTP. The distribution patterns of the formed indol- and catecholamines in a) the pancreatic islets and b) the parafollicular cells were then compared by use of a combined autoradiographic-fluorescence microscopic technique.Localization in the same sections of radioactivity and fluorescence representing the respective monoamines corresponded very well for all the time intervals studied both in the pancreatic islets and in the parafollicular cells.In the pancreatic islets the monoamines were localized in all cells shortly after the injection of the precursors. The monoamines then first disappeared from the -cells leaving the -cells with the highest concentration of activity and fluorescence. In the thyroid the activity and fluorescence, localized in the same parafollicular cells, persisted for a longer time than in the islets.The authors wish to express their gratitude to Professor Sven Ullberg, Head of the Department of Toxicology, University of Uppsala, for much valuable advice during this work. The investigation was supported by grants from the Swedish Medical Research Council (No. 12x-713-01), Knut and Alice Wallenbergs Stiftelse and Ragnar and Torsten Söderbergs Stiftelse.     

Ultrastructural immunocytochemical localization of neuron-specific enolase in parafollicular cells of the rat thyroid

Summary Using pre- and post-embedding procedures, neuron-specific enolase and calcitonin were localized in rat thyroid parafollicular cells by light and electron microscopy. Peroxidase-antiperoxidase (PAP), biotin-avidin (ABC) and protein A — colloidal gold techniques were used. In paraffin sections neuron-specific enolase was demonstrated in all calcitonin-storing parafollicular cells in rats aging 1 to 180 days. The post-embedding procedure failed to detect neuron-specific enolase in ultrathin sections, but the enzyme could be demonstrated using a preembedding procedure. Neuron-specific enolase was localized exclusively within the cytosol of parafollicular cells, while calcitonin was localized within secretory granules applying either post- or pre-embedding incubation techniques.Supported by Sonderforschungsbereich 232     

Calcitonin is not contained within the common precursor to corticotropin and endorphin in the rat.

The suggestion that calcitonin is contained within the structure of the common precursor to ACTH and endorphin was examined. Immunohistochemical staining demonstrated calcitonin in thyroid parafollicular cells, and ACTH and 16K fragment in ACTH/endorphin cells of pituitary. No 16K fragment immunostaining was detected in thyroid parafollicular cells; no calcitonin staining was detected in pituitary. Immunoprecipitation of [³⁵S]methionine-labeled molecules synthesized by rat intermediate pituitary cells demonstrated that neither 30K precursor, 16K fragment nor any other major labeled cell product was recognized by calcitonin antiserum. Analyses of tryptic peptides of 30K precursor indicated that peptides expected from calcitonin were not present in 30K precursor.     

Uptake and storage of indolamines in the ultimobranchial body of the fetal murine thyroid. 1-Autoradiographic histological study]

Incorporation of 3H-5 HTP is studied in ultimobranchial cells and thyroid cells of the mouse foetus from the 13th day to the 18th day of gestation. The APUD characteristics of these cells are first observed on the 14th day when the U.B. bodies are included in the thyroid anlage. The silver granules are then localized on the parafollicular cell cords from which C cells of the adult thyroid arize.     

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.     

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.     

Quantitative electron microscopic autoradiography on the mouse thyroid gland after administration of 3H-L-DOPA

Summary The cellular and subcellular distribution of radioactivity in the mouse thyroid gland different times (20 min — 8 hours) after intravenous administration of ³H-L-DOPA was studied by means of quantitative electron microscopic autoradiography.High concentrations of autoradiographic silver grains occur over parafollicular cells and adrenergic nerves while the labelling of follicular cells and lumina is low or absent and similar to the labelling of connective tissue cells at all observation times.Over the parafollicular cells high levels of radioactivity can be recorded already 20 min after administration of the labelled amino acid. The grain counts are highest at 1 hour and decrease then at 2.5 and 8 hours.The intracellular distribution of label is similar at all observation times; thus, the concentration of silver grains over the typical cytoplasmic granules of the parafollicular cells is 4–5 times higher compared to the concentration over the remainder of the cytoplasm and the nucleus.Treatment with a decarboxylase inhibitor prior to the injection of ³H-L-DOPA results in a low and uniform labelling of all thyroid cells. This finding, taken together with the observation that also pretreatment with reserpine abolishes the autoradiographic reaction over the cytoplasmic granules, gives strong support to the idea that the great majority of silver grains observed over parafollicular cells represents dopamine formed by decarboxylation of the labelled precursor.This study was supported by grant K71-12X-3352-01 from the Swedish Medical Research Council. The author wishes to express his gratitude to Mrs. Gunnel Bokhede and Miss Dala Sjögren for expert technical assistance.     

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.     

Cytological analysis of a population of thyroid parafollicular cells

With the aid of Sevier-Munger silver stain, parafollicular thyrocytes (C-cells) of rat males were investigated within the period of 10 minutes to 8 hours after the intraperitoneal injection of calcium gluconate solution. In the thyroid glands of both control and experimental animals four types of C-cells at different stages of their secretory cycle were described. Relative rates of these cellular types were found to be objective quantitative criteria of the functional activity of parafollicular cell population.     

Monoamines in the C cells of the thyroid gland of callithricid primates

Summary The thyroid gland of three species of marmosets were studied with the Falck and Hillarp fluorescence technique for monoamines. Two types of fluorescent C cells were observed. The predominant type displayed the greenish (or blue) fluorescence characteristic of catecholamines. The other type displayed a yellowish fluorescence most probably due to 5-HT. The presence of monoamines in C cells now reported for the first time in Primates offers an interesting possibility for studying their presumed role in calcitonin secretion.