The effect of chronic hypercalcemia or hypocalcemia on the follicular and parafollicular cells in rat thyroid gland

In three experiments of 30 weeks' duration, 93 adult female Wistar rats received controlled amounts of calcium with food and water, to produce a state of either hypercalcemia or hypocalcemia. A systematic stereological analysis of the thyroid glands and a radioimmunological analysis of thyroxine, triiodothyronine, and thyrotropine were performed. In the hypercalcemic rats, a reactive hyperplasia of the parafollicular cells was established; this was accompanied by morphological and biochemical signs of hyperfunction of the follicular cells, despite a reduced central stimulation by thyrotropin. In the hypocalcemic animals, no quantitative morphological changes in the parafollicular cells were observed; however, morphological and biochemical signs of hypofunction of the follicular cells were obvious, despite stronger central stimulation by thyrotropin. It is concluded that the extrinsic regulation of follicular cells by the blood calcium level is stronger than the intrinsic regulation by hypothalamo-hypophyseal hormones.     

Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O₂) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O₂, 5 h) using the matrigel assay. To further examine the role of HIF-1α in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1α (DNHIF-1α). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1α. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.     

Differential changes in calcitonin,somatostatin and gastrin/cholecystokinin-like immunoreactivities in rat thyroid parafollicular cells during ontogeny

Summary Immunocytochemical quantitative studies on the development of rat thyroid calcitonin (C) cells have been performed. In neonatal rat pups up to day 6 nearly 90% of all calcitonin cells are also somatostatin immunoreactive and 45% of these cells also show immunoreactivity to a C-terminal gastrin/cholecystokinin antiserum (CCK-4-like immunoreactivity). Already at day 8 the frequency of somatostatin immunoreactive calcitonin cells has dropped to 25%, whereas half of the calcitonin cells still display CCK-4-like immunoreactivity. In adult rats, less than 1% of the calcitonin cells are somatostatin immunoreactive, whereas 90% of the calcitonin cells display CCK-4-like immunoreactivity. These data show that between day 6–8 a pronounced change in the peptide repertoire of rat thyroid C cells occur and that these cells, prior to this time, mainly contain calcitonin and somatostatin immunoreactivity and after this time mainly contain calcitonin and CCK-4-like immunoreactivity. The time course of the change in the C cell peptides is similar to that observed with changes in transitory peptides of neonatal rat pancreas and duodenum, suggesting that possible hormonal mechanisms during this period act to change the peptide repertoire of several endocrine cell types simultaneously. It is possible that many of the transitory peptides exert actions in the developing individual that are necessary for growth and differentiation. interestingly, many of these transitory peptides reappear in tumours, where theoretically they could exert similar actions.     

Long-term stimulation of cAMP production in LLC-PK1 pig kidney epithelial cells by salmon calcitonin or a photoactivatable analogue of vasopressin

A photoreactive analogue of vasopressin, [1-(3-mercapto)propionic acid, 8-(N⁶-4-azidophenylamidino)lysine]-vasopressin, was compared to salmon calcitonin and [8-arginine]-vasopressin with respect to stimulation of cAMP synthesis in the LLC-PK₁ pig kidney epithelial cell line. Without photoactivation, the vasopressin analogue-elicited responses were identical to those induced by vasopressin, in that cAMP synthesis returned to the basal, unstimulated level about 4 h after hormonal treatment. In contrast, the levels of activation of cAMP-dependent protein kinase induced by salmon calcitonin returned to basal approx. 12 h after hormone addition. When activated by ultraviolet irradiation, the vasopressin analogue induced ‘permanent’ stimulation of adenylate cyclase, whereby cAMP production could be detected even 12.5 h after treatment. Both salmon calcitonin and the photoactivated vasopressin analogue inhibited growth of LLC-PK₁ cells, in contrast to vasopressin or the nonactivated analogue. Growth inhibition appeared to be a consequence of the prolonged stimulation of adenylate cyclase. This conclusion was supported by the fact that a LLC-PK₁ cell mutant in cAMP-dependent protein kinase was resistant to growth inhibition by salmon calcitonin and activated vasopressin analogue. The results imply that the cAMP-dependent protein kinase is the mediator of the hormone-stimulated growth inhibition.     

Overexpression and purification of recombinant eel calcitonin and its phylogenetic analysis

Calcitonin (CT), a peptide hormone that is widely used for the treatment of osteoporosis, Paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients, is produced by the para-follicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Fish calcitonin, like eel calcitonin (eCT), is more potent and longer lasting than human CT and is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated eCT in recombinant Streptomyces avermitilis. A phylogenetic analysis was performed with all the known CT amino acid sequences.     

The mechanisms by which vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) stimulate prolactin release from pituitary cells

The effect of vasoactive intestinal peptide (VIP) on prolactin (PRL) secretion from pituitary cells is reviewed and compared to the effect of thyrotropin releasing hormone (TRH). These two peptides induced different secretion profiles from parafused lactotrophs in culture. TRH was found to increase PRL secretion within 4 s and induced a biphasic secretion pattern, while VIP induced a monophasic secretion pattern after a lag time of 45–60 s.The secretion profiles are compared to changes in adenylate cyclase activity, production of inositol polyphosphates, changes in intracellular calcium concentrations and changes in electrophysiological properties of the cell membrane.Abbreviations AC adenylate cyclase - DG diacyglycerol - GH growth hormone - GTP guanosine trisphosphate - G_i GTP binding proteins that mediate inhibition of adenylate cyclase and that are pertussis toxin sensitive - G_s GTP binding protein that mediates stimulation of adenylate cyclase - GH cells clonal rat pituitary tumor cells producing PRL and/or growth hormone - GH₃ GH₄C₁ and GH₄B6 subclones of GH cells - PKA protein kinase A - PKC protein kinase C - PLC phospholipase C - PRL prolactin - TPA 12-O-tetradecanoyl phorbol 13-acetate - TRH thyrotropin releasing hormone - VIP vasoactive intestinal peptide     

Depletion of secretory granules,calcitonin, and formaldehyde-ozone-induced fluorescence from cat thyroid C cells by vitamin D2 treatment

Summary Using a combination of electron microscopy, fluorometry, and bioassay, the C cells of the cat thyroid were investigated with respect to their content of secretory granules, and calcitonin, and to their formaldehyde-ozone-induced fluorescence. This fluorescence is assumed to reflect the presence of a peptide with NH₂-terminal tryptophan. In cats injected with large doses of vitamin D₂ daily for 5 days, the C cells were degranulated, their fluorescence intensity was lowered and the calcitonin content of the thyroid was markedly reduced. It is suggested that the proposed tryptophyl peptide in the C cells is stored in the secretory granules and that it is engaged in the storage and/or release of the hormone.     

Differential changes in calcitonin, somatostatin and gastrin/cholecystokinin-like immunoreactivities in rat thyroid parafollicular cells during ontogeny

Immunocytochemical quantitative studies on the development of rat thyroid calcitonin (C) cells have been performed. In neonatal rat pups up to day 6 nearly 90% of all calcitonin cells are also somatostatin immunoreactive and 45% of these cells also show immunoreactivity to a C-terminal gastrin/cholecystokinin antiserum (CCK-4-like immunoreactivity). Already at day 8 the frequency of somatostatin immunoreactive calcitonin cells has dropped to 25%, whereas half of the calcitonin cells still display CCK-4-like immunoreactivity. In adult rats, less than 1% of the calcitonin cells are somatostatin immunoreactive, whereas 90% of the calcitonin cells display CCK-4-like immunoreactivity. These data show that between day 6-8 a pronounced change in the peptide repertoire of rat thyroid C cells occur and that these cells, prior to this time, mainly contain calcitonin and somatostatin immunoreactivity and after this time mainly contain calcitonin and CCK-4-like immunoreactivity. The time course of the change in the C cell peptides is similar to that observed with changes in transitory peptides of neonatal rat pancreas and duodenum, suggesting that possible hormonal mechanisms during this period act to change the peptide repertoire of several endocrine cell types simultaneously. It is possible that many of the transitory peptides exert actions in the developing individual that are necessary for growth and differentiation. Interestingly, many of these transitory peptides reappear in tumours, where theoretically they could exert similar actions.     

Basal and stimulated calcitonin secretion in primary hyperparathyroidism before and after parathyroidectomy

To investigate calcitonin secretion in primary hyperparathyroidism, basal and stimulated (3 mg Ca++/kg body weight/10 min) immunoreactive calcitonin plasma levels were studied before parathyroidectomy. Plasma calcitonin levels were raised in 50% of patients regardless of sex, but a significant correlation between basal plasma calcium and calcitonin was found only in males. A reduced calcitonin response to calcium infusion was observed in all patients. Parathyroidectomy invariably induced a normalization of calcitonin basal levels. Our findings confirm the existence of a decreased parafollicular cell reserve probably as a consequence of the persistent hypercalcemic state in hyperparathyroid patients and suggest that it is more frequent in females.     

Effect of vitamin D2 stimulation on amine stores and secretory granules in the calcitonin cells of the mouse

Summary The calcitonin-producing cells (C cells) of the thyroid gland are characterized by numerous suhmicroscopic granules, believed to contain the hormone, by a strong argyrophilia, and by the ability to synthesize and store certain arylethylamines (such as dopamine) from corresponding immediate precursors (such as DOPA).In mice, vitamin D₂ treatment—known to increase calcitonin secretion—evoked a degranulation and a loss of argyrophilia of the C cells, and a reduction in their content of dopamine (induced by exogenous DOPA). Additional treatment with a monoamine oxidase inhibitor restored the dopamine content of the C cells, although they were still degranulated. Treatment with reserpine, which inhibits the amine-storing mechanism, prevented the degranulation following vitamin D₂ treatment.The findings support the assumption that a functional rather than only a structural relationship exists between the intracellular amine and the secretory granules in cells elaborating calcitonin, and that the amine is involved in the secretion of the polypeptide hormone.     

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.     

Pituitary adenylate cyclase-activating peptide (PACAP) recruits low voltage-activated T-type calcium influx under acute sympathetic stimulation in mouse adrenal chromaffin cells

Low voltage-activated T-type Ca_v3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Ca_v3.2-dependent conductance. However, Ca_v3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Ca_v3.2 exists, there are no reports testing the specific role of Ca_v3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Ca_v3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca²⁺-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Ca_v3.2 Ca²⁺ influx. This in turn evokes catecholamine release during the acute sympathetic stress response.     

Morphofunctional characteristics of the recovery processes in the resected thyroid of rats

Histological, electron microscopy and radiometry methods were used to study the time-course of the recovery of the morphological and functional parameters of the rat thyroid after resecting 2/3 of the organ. The signs of thyrocyte regeneration, namely hypertrophy of the cells and ultrastructures, proliferation and formation of new follicles were most pronounced on the 5th day. The increase in the number of C cells, their hypertrophy and proliferation permit attaining the high calcitonin level in the blood serum, reaching a maximum on the 15th experimental day.     

Calcitonin receptors in a cloned human breast cancer cell line (MCF 7)

A human breast cancer cell line, MCF 7, is shown to possess a specific calcitonin receptor and calcitonin responsive adenylate cyclase, and calcitonin treatment results in activation of cyclic AMP-dependent protein kinase. Studies with several analogues of calcitonin show that the receptor and adenylate cyclase response preserve the ability to discriminate among the structure-function relationships of the calcitonin molecule. The same cell line has been shown recently to possess a receptor for the steroid hormone, 1,25(OH)₂-vitamin D. Coexistence in MCF 7 cells of receptors for two calcium-regulating hormones may be related to the osteoclast-like properties of these cells.     

Pulmonary immunoreactive calcitonin in the African green monkey (Cercopithecus aethiops): Anatomic distribution

The hormone calcitonin, which occurs predominantly within the C cells of the mammalian thyroid gland, is also found within the pulmonary endocrine cells of the epithelium of the tracheobronchial tree. A study was made of the distribution of immunoreactive calcitonin (iCT) in the African green monkey. Using two different region-specific antisera, the total respiratory iCT comprised 2.5% and 5.8% of the total thyroid iCT. The mean concentration of iCT in the right lung exceeded that in the left, and the mean concentration of the right middle or right upper lobe exceeded that of all other lobes. Embryologically, the ultimobranchial bodies contribute their iCT-producing C cell primordia to the thyroid gland near the level of the primitive laryngotracheal cleft and shortly after the early arborization of the bronchial tree. In monkeys and most other mammals, the right main stem bronchus is larger and develops earlier than the left. The data suggest an early migration of cells from the ultimobranchial bodies to the bronchi, eventually giving rise to the iCT-containing pulmonary endocrine cells.     

Morphological changes in the ultrastructure of the thyroid epithelium of Notophthalmus viridescens after hypophysectomy and TSH- and prolactin stimulation

Summary In the newt, Notophthalmus viridescens, hypophysectomy results in progressive atrophy of the thyroid cells to the point of irreversible degeneration. After exclusive TSH-stimulation of hypophysectomized newts, increased endocytotic activity of the follicular epithelium is observed. Prolactin stimulation under the same conditions prevents atrophy but does not result in increased cell activity, as expressed by the reduced amount of microvilli and the lack of endocytotic activity. Combined TSH- and prolactin stimulation also results in cell activation, but the activation level of exclusively TSH-stimulated cells is not reached. Although prolactin prevents cellular atrophy and degeneration of the follicular epithelium, it reduces the TSH-induced activation of the thyroid epithelium.     

Overexpression, purification and characterization of recombinant salmon calcitonin, a therapeutic protein, in Streptomyces avermitilis

Calcitonin (CT) is a peptide hormone produced by the parafollicular cells of the thyroid gland in mammals and by the ultimobranchial gland of birds and fish. Salmon calcitonin (sCT), which is more potent and longer lasting than human CT, has been used widely for the treatment of osteoporosis, paget's disease, hypercalcemic shock and chronic pain in terminal cancer patients. sCT is one of the many bioactive peptides that require C-terminal amidation for full biological activity. In this study we describe the over-expression and over-production of C-terminal amidated sCT in recombinant Streptomyces avermitilis. With this approach the utilization of expensive peptide synthesis can be circumvented.