Compensatory-adaptive modifications of the C-cell apparatus of the thyroid in normal rats of various ages and in partial sympathectomy

Population of the thyroid C-cells, normal and at sympathectomy has been analyzed in 75 white male rats at the age of 1, 3 and 6 months by means of electron microscopical, morphometrical and radioimmunological methods. Partial sympathectomy has been performed using subcutaneous injection of guanethidine (15 mg/kg of body mass) during 14 days after birth. During the period from 1 up to 6 months of life in intact rats a decrease in C-cells functional activity is observed. Under conditions of sympathectomy in 30-day-old animals decreasing extrusion processes of the secretory material are observed. During successive periods of life (3 and 6 months) mechanisms of paracrinic evacuation of hormonal products enhance considerably, nuclear volume of the cells and number of C-cells in the field of vision increase. Their hyperplastic alterations in the sympathectomized thyroid gland are more pronounced in 3-month-old animals.     

Quantitative changes of the C-cell population in the rat thyroid during postnatal ontogenesis

Summary The number and distribution of C-cells in the rat thyroid gland, have been investigated during postnatal ontogenesis from birth to 120 days of age. The argyrophilic and metachromatic properties of these cells were used to identify them. In the thyroid of newborn rats the C-cells do not exhibit argyrophilia and metachromasia. These reactions appear at 10 days and can be seen at all subsequent ages. The number of C-cells shows a parallel increase with age as demonstrated by the change in the proportion of C-cellsF-cellscolloidstroma during development. A marked increase in C-cells was found at 50 days of age when the proportion of C-cells rose to 27.67% from the value of 16.78% at 30 days. At 70 days a decrease was noted (20.50%) which hardly changed until 120 days of age (22.20%). The numerical increase in C-cells occurs at the expense of the follicular epithelium and stroma.The C-cells occupy elongated islet-like region in the central part of the lobe, decreasing in number towards the periphery where no C-cells are present. The long axis of the C-cells area is parallel with the longitudinal axis of the lobe. The area of C-cells is largest at the centre of the lobe, corresponding to the territory of the peak of the Gaussian curve for the numerical distribution of C-cells.     

Thyroid C cell function during fasting and refeeding of young and old rats

Age-related alterations in the structure and function of many organs often become apparent under stimulation of their function. Although the ageing process affects the regulation of mineral homeostasis, the function of thyroid C-cells that secrete calcitonin (CT) under the conditions of fasting and refeeding, a way of dietary manipulation that reveal the existence of age-related changes of follicular thyroid cells, has not been characterized. Therefore, we investigated the number of C-cells and serum CT concentration in young (4 mo) and old (26 mo) male rats fasted for 48 hours, and then refed for 4 or 24 hours. We found significantly higher number of C-cells in thyroids of old vs young rats both under basal conditions, and after fasting/refeeding. Correspondingly, serum calcitonin level was higher in fed or fasted old rats vs young ones. However, in young rats refeeding decreased, whereas in old animals increased serum concentrations of calcitonin. Thus, the control of serum calcium concentration, that was well preserved in old rats, occurs at the expense of increased serum CT level both under basal conditions, and after refeeding. These observations suggest that C-cell function is altered in ageing.     

Postnatal variations in the number and size of C-cells in the rat thyroid gland

The heterogeneous distribution of thyroid C-cells has until now hindered an objective evaluation of changes caused by age or experimental stimuli. To overcome this, a rigorous methodology has been designed to detect variations in shape, size, and number of C-cells throughout development. Using this methodology, we have demonstrated that C-cells do not significantly alter their shape with age. However, their volume increases gradually from 472 m³ in newborn rats to 1653 m³ in 120-day-old animals. Over the same time period, the mean number of C-cells within the thyroid gland increased 9-fold (from 1.6x10⁴ to 1.5x10⁵), and the number of C-cells per unit area decreased (from 6.15x10⁴/mm³ to 2.6x10⁴/mm³). We conclude that there are marked variations in size, total number, and number of C-cells per unit area in the rat thyroid gland after birth.     

Production of monoclonal antibodies against a novel glycoprotein synthesized and secreted by dog thyroid C-cells.

A monoclonal antibody (MAb) that reacted only with thyroid C-cells was raised against cell suspensions from dog thyroid glands, to examine a glycoprotein secreted by C-cells. After chronically-induced hypercalcemia and administration of an anti-thyroid drug, reaction products for the antibody markedly decreased in C-cells, coinciding with alterations in calcitonin immunoreactivity. The antigen recognized by the MAb appears to be a secretory protein. The MAb reacted with C-cells from a wide variety of mammalian species, including rats, mice, hamsters, cattle, cats, rabbits, and monkeys. Furthermore, tumor cells of human medullary thyroid carcinoma, which is derived from C-cells, were immunoreactive to the MAb. Exceptionally, C-cells from guinea pigs and pigs were not stained with the MAb. No crossreactivity was observed in any of the dog tissues examined. Immunoblot analysis demonstrated that the MAb recognized a single prominent band at a molecular weight of approximately 79,000. The 79 KD band reacted with various digoxigenin-labeled lectins, including GNA, DSA, SNA, and MAA; it is a glycoprotein containing mannose, N-acetylglucosamine, and sialic acid. Dog thyroid C-cells were also densely stained with these lectins. The results indicate that thyroid C-cells synthesize and secrete a specific glycoprotein in addition to peptide hormones.     

Decrease in calcitonin and parathyroid hormone mRNA levels and hormone secretion under long-term hypervitaminosis D3 in rats

In calcium homeostasis, vitamin D3 is a potent serum calcium-raising agent which in vivo regulates both calcitonin (CT) and parathyroid hormone (PTH) gene expression. Serum calcium is the major secretagogue for CT, a hormone product whose biosynthesis is the main biological activity of thyroid C-cells. Taking advantage of this regulatory mechanism, long-term vitamin D3-induced hypercalcemia has been extensively used as a model to produce hyperactivation, hyperplasia and even proliferative lesions of C-cells, supposedly to reduce the sustained high calcium serum concentrations. We have recently demonstrated that CT serum levels did not rise after long-term hypervitaminosis D3. Moreover, C-cells did not have a proliferative response, rather a decrease in CT-producing C-cell number was observed. In order to confirm the inhibitory effect of vitamin D3 on C-cells, Wistar rats were administered vitamin D3 chronically (25,000 IU/d) with or without calcium chloride (CaCl2). Under these long-term vitamin D3-hypercalcemic conditions, calcium, active metabolites of vitamin D3, CT and PTH serum concentrations were determined by RIA; CT and PTH mRNA levels were analysed by Northern blot and in situ hybridization; and, finally, the ultrastructure of calciotrophic hormone-producing cells was analysed by electron microscopy. Our results show, that, in rats, long term administration of vitamin D3 results in a decrease in hormone biosynthetic activities of both PTH and CT-producing cells, albeit at different magnitudes. Based upon these results, we conclude that hypervitaminosis D3-based methods do not stimulate C-cell activity and can not be used to induce proliferative lesions of calcitonin-producing cells.     

Quantitative changes in the frequency and distribution of the C-cell population in the rat thyroid gland with age

Summary The development of calcitonin cells (C-cells) was investigated in rat thyroid glands from birth to 120 days, using an immunoperoxidase technique and a point-counting method. The proportion of C-cells to follicular cells was 4.5% on the day of birth and increased progressively to 10.4% by 120 days. The highest density of C-cells was noted in the mid-region of the lobes along a longitudinal axis. The caudal and cephalic regions of the lobes contained smaller numbers of C-cells. The C-cells tended to be more numerous in the posterior aspects of the lobes. Although the numbers of C-cells in 120-day-old animals were markedly increased as compared to animals at the time of birth, the cell distributions within the glands were similar at all ages.     

Survival in foods of Staphylococcus aureus grown under optimal and stressed conditions and the effect of some food preservatives

Staphylococcus aureus was grown in a rich peptone medium which became alkaline with continued incubation. Cells were grown at 37 degrees C and in the same medium containing 1 M NaCl at 46 degrees C, a temperature at which this organism can grow only when protected by NaCl. Cells of these cultures are hereafter called 37 degrees C-cells and 46 degrees C-cells, respectively. The 37 degrees C-cells harvested when the pH was 7.1 to 7.7 had decimal reduction times (D60-value) of 1.8 to 3.1 min in 50 mM pH 7.2 Tris buffer. The D60 value of 46 degrees C-cells tested in the same way, harvested from cultures at pH 6.6 to 7.6, ranged from 5.3 to a maximum of 12.8 min. In milk, green beans, peas, or beef slurry, the D60-value of 46 degrees C-cells was about four times higher than that of 37 degrees C-cells. Length of survival after freeze-drying in skim-milk powder exposed to air was longest for the cells with the highest D-value. In freeze-dried peas and media acidified with acetic and lactic acids, 46 degrees C-cells survived longer than 37 degrees C-cells. However, the sensitivity of the two kinds of cells to potassium sorbate, sodium benzoate, and sodium propionate was essentially the same, but the 46 degrees C-cells were more resistant to butylated hydroxyanisole and sodium nitrite.     

Histometry of normal thyroid glands in neonatal and adult rats

The present histometric study is on thyroid glands of Wistar rats ranging in age from 0 to 120 days. The mean volume of one lobe of the thyroid in 4-month-old animals was some 22-, 10-, 5-, and 3-fold greater, respectively, than the volumes in the newborn, 5-, 10-, and 30-day-old rats. At 4 months of age the mean length of the lobe was 3 times greater than at birth. The volumetric fractions (Vv) of the different histological components (follicular cells, C-cells, colloid, and interstitial tissue) changed considerably in the course of development. The Vv of follicular cells diminished from 61.4% at birth to 37.2% at 4 months. C-cells increased from 2.9% in the newborn to 4% at 15 days, with no further significant change at 4 months. Colloid and stroma together represented 35.7% at birth, increasing to 58.5% at 120 days. In the course of the first 4 months of life, the absolute volumes occupied by follicular cells, C-cells, colloid, and stroma increased 13.25, 30.75, 38.6, and 33.7 times, respectively; these changes reflect the variations that occur in the volume of the gland and the Vv throughout postnatal development.     

Adrenal catecholamine synthesis rate changes induced by combined thermal and immobilization stress in fed and 24 hour fasted rats

The combined stress of acute immobilization (IM) at high and low ambient temperature has been used to determine its influence on adrenal catecholamine (CA) content assassed histofluorimetrically in fed and 24 hour fasted rats. The general course of changes obtained after the arrangement of adrenal strips deriving from the adrenals of rats exposed to cold and IM stress (CIMS) at +10 degrees C to -25 degrees C during the different time fragments presented the adrenal CA depletion events followed sometimes by the adrenal CA content increase after the longer stress exposure or/and stronger CIMS and WIMS conditions. It was found that this depletion-stimulated increase of adrenal Ca synthesis rate had been accelerated in 24 h fasted rats compared to satiated ones exposed to the same stress conditions, especially after the CIMS exposure. Moreover the survival time duration at first lethal temperature (-5 degrees C and +45 degrees C) was significantly higher in fasted rats. The possible hypothalamic regulation of adrenal CA synthesis rate accordingly to the actual metabolism needs and beta-adrenoceptor sensitivity changes depending on satiety state have been discussed and the necessity of further investigations concerning the specificity of stress-induced metabolism changes in 24 h starved rats has been suggested.     

Thyrotropin-releasing hormone receptor expression in thyroid follicular cells: a new paracrine role of C-cells?

Thyrotropin-releasing hormone (TRH) synthesized in the hypothalamus has the capability of inducing the release of thyroid-stimulating hormone (TSH) from the anterior pituitary, which in turn stimulates the production of thyroid hormones in the thyroid gland. Immunoreactivity for TRH and TRH-like peptides has been found in some tissues outside the nervous system, including thyroid. It has been demonstrated that thyroid C-cells express authentic TRH, affecting thyroid hormone secretion by follicular cells. Therefore, C-cells could have a paracrine role in thyroid homeostasis. If this hypothesis is true, follicular cells should express TRH receptors (TRH-Rs) for the paracrine modulation carried out by C-cells. In order to elucidate whether or not C-cell TRH production could act over follicular cells modulating thyroid function, we studied TRH-Rs expression in PC C13 follicular cells from rat thyroid, by means of immunofluorescence technique and RT-PCR analysis. We also investigated the possibility that C-cells present TRH-Rs for the autocrine control of its own TRH production. Our results showed consistent expression for both receptors, TRH-R1 and TRH-R2, in 6-23 C-cells, and only for TRH-R2 in PC C13 follicular cells. Our data provide new evidence for a novel intrathyroidal regulatory pathway of thyroid hormone secretion via paracrine/autocrine TRH signaling.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Structural organization of the adrenal cortex in rats with different types of autonomic reactivity under standard conditions and after exposure to different altitudes

The structural organization of adrenal cortex has been studied in intact rats and after short-term hypoxia exposure on different altitudes (2, 6, 8, 11 km). Previously rats have been divided into 3 groups by adrenaline test for determining of vegetative reactivity type. It has been found that some histochemical and morphometric indices of adrenal cortex had peculiarities with respect to type of vegetative reactivity (width of zones, their ratio, nuclear diameter, ascorbic acid content and its distribution and other) in intact rats. Under altitude these differences form individual character of steroidogenesis and determine resistance and reactivity of organism.     

Diurnal Rhythms of Rat Liver 3-Hydroxy-3-Methylglutaryl-Co A Reductase and D Site-Binding Protein mRNA Levels Are Not Abolished by Adrenalectomy

The effect of glucocorticoids on the diurnal rhythm of rat liver 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) has been controversial. Also, diurnal variation of D site-binding protein (DBP) has been suggested to be under a negative control of glucocorticoids. Here we have re-evaluated the effects of adrenal hormones on these rhythms at the level of gene expression. Sham-operated and bilaterally adrenalectomized rats were killed at 4-hr intervals and total RNA from each liver was subjected to Northern blot analysis. Diurnal variation patterns of HMGR and DBP mRNA levels in adrenalectomized rats were substantially identical to those in sham-operated rats, although DBP mRNA levels in adrenalectomized rats were slightly more abundant than in control rats. HMGR mRNA levels in adrenalectomized rats in the dark period were insensitive to a single injection of adrenal hormones, whereas the augmented levels of DBP mRNA in these animals were returned to the control levels by this treatment, indicating that glucocorticoids are prone to decrease the amplitude of variation in the DBP gene expression. The present results suggest that adrenal hormones are not critical for the generation of diurnal rhythms of these mRNAs.     

Light and electron microscopic evidences of the presence of c-fos-like immunoreactivity in the rat adrenal cortex

Summary The presence and localization of c-fos-like immunoreactivity in the rat adrenal cortex has been demonstrated by immunocytochemical methods at both light and electron microscopic level. C-fos-like immunoreactivity was detected in the zona fasciculata and the zona reticulata, but not in the zona glomerulosa. Ultrastructurally, all products of c-fos-like immunoreaction were localized exclusively in the regions associated with the euchromatin in the nucleus of the immunoreactive cells. Moreover, a higher density of the immunoreactive cells in the adrenal cortex of pregnant rats was found with quantitative immunocytochemistry as compared to the non-pregnant. The characteristic zonation of c-fos-like immunoreactivity in the adrenal cortex suggest that the c-fos protein is involved in the normal function of the glucocorticoid-producing cells of mammalian adrenals. The numerical increase in the immunoreactive cells in pregnant rats implies that basal expression of the c-fos-like protein may vary with the functional state of the cortical cells.     

Effect of ethanol on corticosterone production by dispersed adrenal cells of the rat

The action of ethanol on adrenal steroidogenesis "in vitro" has been studied. It has been found that ethanol did not change the basal production of corticosterone by dispersed adrenal cells, but significantly reduced its response to ACTH stimulation. It is suggested that the inhibitory action of ethanol on steroidogenesis "in vitro" could have a physiological meaning, because the response to ACTH stimulation of adrenal cells from rats treated "in vivo" with ethanol showed a clear dose-related inhibition.     

Light and electron microscopic evidences of the presence of c-fos-like immunoreactivity in the rat adrenal cortex

The presence and localization of c-fos-like immunoreactivity in the rat adrenal cortex has been demonstrated by immunocytochemical methods at both light and electron microscopic level. C-fos-like immunoreactivity was detected in the zona fasciculata and the zona reticulata, but not in the zona glomerulosa. Ultrastructurally, all products of c-fos-like immunoreaction were localized exclusively in the regions associated with the euchromatin in the nucleus of the immunoreactive cells. Moreover, a higher density of the immunoreactive cells in the adrenal cortex of pregnant rats was found with quantitative immunocytochemistry as compared to the non-pregnant. The characteristic zonation of c-fos-like immunoreactivity in the adrenal cortex suggest that the c-fos protein is involved in the normal function of the glucocorticoid-producing cells of mammalian adrenals. The numerical increase in the immunoreactive cells in pregnant rats implies that basal expression of the c-fos-like protein may vary with the functional state of the cortical cells.     

The Use of 1:9-Dimethygmethylene Blue for the Demonstration of Thyroid C-Cells

1:9-dimethyl-methylene blue was used for the demonstration of C-cells in the thyroid of dog and rat. After hydrolysis with HCI at 60 C selective staining of C-cells was obtained. The metachromatic reaction was intense, and increased in stability if ammonium heptamolybdate was applied.     

Anomalous occurrence of immunoreactive calcitonin cells in the thymus of the rat

Summary In a study of the effect of pinealectomy on thyroid C-cell number, 8 animals out of 66 were found to have thymic tissue in close association with the thyroid. Cells containing immunoreactive calcitonin were found in all of the thyroids but in only one of the 8 pieces of thymus. These cells found in a piece of thymic tissue associated with the right thyroid lobe were located immediately under the capsule and did not form or associate with follicles. Unlike the other animals the rat with thymic calcitonin cells had an unequal distribution of C-cells between the left and right thyroid lobes, but the total number of thyroidal C-cells was the same as that of the other rats. Since the thymus proper was not examined in these 66 animals, ten additional rats were taken for such a study. Thyroid-associated thymic tissue was found in three of these, but none of these thymi showed any immunoreactive cells.Financial support by the Deutsche Forschungsgemeinschaft (grant Vol 35/7) is gratefully acknowledged     

Cholecystokinins but not gastrin-17 release calcitonin from thyroid C-cells in the rat

Subcutaneous injections of gastrin-17, cholecystokinin-39, cholecystokinin-8 (sulfated and non-sulfated forms), cholecystokinin-4 or pentagastrin induced hypocalcemia in rats. The hypocalcemia was associated with calcitonin release for pentagastrin and the cholecystokinins but not for gastrin-17, even at very high doses. Permanent hypergastrinemia, induced by surgical removal of the acid-producing part of the stomach (fundectomy) or by treatment with high doses of omeprazole, a blocker of acid secretion, was not accompanied by elevated plasma calcitonin. Long-lasting hypergastrinemia is known to cause hyperplasia of gastrin-sensitive endocrine cells in the rat stomach while hypogastrinemia does the reverse. In antrectomized rats, having low serum gastrin, and in fundectomized rats, having high serum gastrin, the serum calcitonin concentration, the thyroid calcitonin content and the number of C-cells remained as in sham-operated controls two months after the operations. We conclude that neither exogenous nor endogenous gastrin stimulates calcitonin secretion in the rat and that long-standing hypo- or hypergastrinemia is without effect on the number of thyroid C-cells. Our results, however, do not exclude the possibility that the cholecystokinins might act as calcitonin secretagogues in the rat although such a role remains to be established.