Biomedical subjects

J F Denef

Publications and source records attributed to J F Denef.

At least 37 records · Page 2Linked to original sources

PubMed1995

Site-dependent pathological differences in smooth muscles and skeletal muscles of the adult mdx mouse.

This study presents a survey of the morphometric characteristics, the regeneration rate, and the extent of muscle dystrophy in several smooth and skeletal muscles from adult mdx mice, an animal model of the Duchenne muscular dystrophy (DMD). Smooth muscles from adult mdx mice showed neither cell necrosis nor fibrosis. As compared to control C57 mice, the thickness of the mdx smooth muscle was normal in the vascular and urogenital layers but significantly reduced in the digestive layers, a finding relevant to clinical reports of gastrointestinal dilatation in DMD patients, and suggesting that gastrointestinal dysfunctions should be systemically searched for in DMD patients. Adult mdx skeletal muscles, however, presented different patterns of muscle suffering: either absent (esophagus); very mild (trunk and limb muscles); or severe (diaphragm). In these three conditions we studied the fiber diameters, the nuclei locations, and the regeneration rate. From this comparative study, it seems that severe dystrophy occurs in muscle tissues showing large fiber diameter and peripheral location of the nuclei. We showed that this combination occurs in the mouse diaphragm which is thus a realistic model for human DMD muscles.

Animals↗

PubMed1995

Sex-steroid control of galanin in the rat hypothalamic-pituitary axis.

To further investigate how sex steroids regulate galanin (GAL) in the rat pituitary and hypothalamus, we examined the effects of prepubertal gonadectomy (Gx) and long-term (9 weeks) replacement with estradiol (E2) or testosterone (T) on pituitary and hypothalamic GAL concentrations in Wistar rats (5-6/group). Sham-operated animals served as controls (CTR). Pituitary GAL concentration was markedly higher in random-cycling CTR-females than in CTR-males (1391 +/- 247 vs 39 +/- 5 pg/mg protein, P < 0.01) and decreased after Gx only in females (20 +/- 3 pg/mg protein, P < 0.01). E2 strongly increased pituitary GAL in Gx-females and Gx-males (4470 +/- 365 and 3853 +/- 347 pg/mg protein, P < 0.01), whereas T had no effect. Inversely, hypothalamic GAL was higher in CTR males than in CTR females (5.4 +/- 0.3 vs 4.0 +/- 0.5 ng/mg protein, P < 0.05), and decreased significantly after gonadectomy in males (3.7 +/- 0.2 ng/mg protein, P < 0.01). The only steroid treatment that significantly modified hypothalamic GAL in Gx animals was administration of E2 to females (5.7 +/- 0.4 ng/mg protein, P < 0.01 vs non-treated Gx). We also studied in hypophysectomized (Hx) rats (8/group) the effects of sex steroids on hypothalamic GAL concentration and distribution. The low hypothalamic GAL concentration observed in male and female Hx rats (1.0 +/- 0.1 ng/mg protein) was significantly increased by T in males and in females (respectively, by 40% and by 50%, P < 0.02) and by E2 in males (by 60%, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Expression of nitric oxide synthase isoforms in the thyroid gland: evidence for a role of nitric oxide in vascular control during goiter formation.

The thyroid gland is a highly vascular tissue, and its blood flow changes dramatically in various pathological conditions. Although the mechanisms regulating these changes in vascularity and blood flow are not well understood, candidate mediators include endothelin-1 (ET-1) and nitric oxide (NO). In the present study, we used a reverse transcriptase-polymerase chain reaction assay to determine which components of these vasoregulatory pathways are present in the thyroid and to analyze changes in gene expression in an experimental model of goiter formation and involution. Expression of messenger RNAs (mRNAs) encoding ET-1, ET receptors (ETA and ETB), ET-converting enzyme, and the three nitric oxide synthase (NOS) isoforms (NOS I, NOS II, and NOS III) was readily detected in the rat thyroid. After goiter formation was induced by thiouracil and a low iodine diet, there was increased expression of the genes encoding ET-related proteins (ET-1, 3.2-fold; ETA, 2.9-fold; ETB, 3.5-fold) as well as two of the three NOS isoforms (NOS I, 2.7-fold; NOS III, 4.9-fold). During iodide-induced involution, the ET-related mRNA levels remained elevated, whereas those of the two NOS isoforms returned to basal values. ET-converting enzyme, NOS II, and thyroglobulin mRNAs were minimally affected in this model, providing evidence for selective regulation of these genes. To assess whether NO plays a role in vascular changes during goiter formation, animals were treated with a NOS inhibitor, N-nitro-L-arginine methyl ester (NAME). NOS activity in the thyroid was inhibited by more than 75% after treatment with NAME. Thyroid hormone and TSH levels were unchanged. Although NAME had little effect on overall thyroid size, vascular expansion during goiter formation was decreased by 36%. We conclude that the thyroid gland expresses a complex network of vasoactive genes whose expression is regulated dynamically during thyroid goiter formation and involution. NO production and probably other locally produced vasoactive substances are involved in changes in thyroid vascularization.

Animals↗

PubMed1995

Effects of selenium deficiency on thyroid necrosis, fibrosis and proliferation: a possible role in myxoedematous cretinism.

It has been suggested that selenium deficiency is a co-factor to iodine deficiency in the pathogenesis of myxoedematous cretinism. The mechanism proposed is that the generation of hydrogen peroxide is greatly increased in iodine-deficient thyroid glands, and that selenium is involved in the control of hydrogen peroxide and its derived free radicals. This study was carried out to investigate the effect of the possibly impaired cellular defence mechanism associated with selenium deficiency on thyroid necrosis and tissue repair. For this purpose, we studied thyroid tissue from selenium- (SE-) and/or iodine-deficient (I-) rats before and after an acute toxic iodine overload. In I- thyroids, necrotic cells were numerous. Acute iodine administration increased this effect. Necrosis was associated with transient infiltration of inflammatory cells. In I-SE+ thyroids the tissue resumed its normal appearance. In I-SE- thyroid glands, the iodide toxicity was stronger, with greater necrosis and inflammatory reaction. The inflammation resolved but was replaced by fibrotic tissue. Fifteen days after the toxic overload, the connective tissue volume was twice the control value. Before iodide overload, the proportion of dividing cells was equal in I-SE+ and I-SE- thyroids. Three days after the iodide overload, this proportion was increased in I-SE+ thyroids but reduced in the I-SE- thyroids. Overall, the I-SE- thyroids had four times fewer dividing cells than the I-SE+ thyroids. In summary, selenium deficiency coupled to iodine deficiency increased necrosis, induced fibrosis and impeded compensatory epithelial cell proliferation. These results are compatible with histological and functional description of thyroid tissue from myxoedematous cretins.

Animals↗

PubMed1995

Two-step development of Hashimoto-like thyroiditis in genetically autoimmune prone non-obese diabetic mice: effects of iodine-induced cell necrosis.

The administration of a high iodide dose (HID; 10 micrograms/day) to goitrous mice is known to induce thyroid cell necrosis and inflammation, which, in most strains, is transient. In this study, we analyzed the effects of iodide in autoimmune prone non-obese diabetic (NOD) mice. Control NOD mice fed a standard diet (MID; 1 microgram I/day) or HID did not spontaneously develop thyroiditis. In NOD mice previously made goitrous, HID provoked thyroid cell necrosis and diffuse inflammation within 4 days. Inflammatory cells consisted of MHC-class II+ antigen-presenting cells, CD4+ T helper cells and CD8+ T suppressor/cytotoxic cells. After 96 days of treatment with HID, thyroiditis similar to Hashimoto's disease was obtained in 100% of the animals, with destruction of thyroid follicles, large clusters of T and B cells, and antithyroid antibodies in the plasma. When treating goitrous mice with MID, no cell necrosis was observed and no autoimmune thyroiditis was obtained. The early iodide-induced cell necrosis and inflammation may thus be considered as an important factor in the induction and persistence of autoimmune thyroiditis in individuals carrying a genetic susceptibility to autoimmune disease.

Animals↗

PubMed1994

Subtypes of Madin-Darby canine kidney (MDCK) cells defined by immunocytochemistry: further evidence for properties of renal collecting duct cells.

The Madin-Darby canine kidney (MDCK) cell line has been proposed as a model for studying intercalated (IC) cells of the renal cortical collecting duct. The IC cells are characterized by peanut lectin (PNA) binding capacity, carbonic anhydrase (CA) activity and Cl(-)-HCO3- exchange mediated by a band 3-related protein. It has been suggested that these properties are also expressed in MDCK cells. So far however, the nature of the specific protein involved in Cl(-)-HCO3- exchange, the type of CA isozyme and the relationship between these two characteristics and PNA binding, have not been investigated in MDCK cells by immunocytochemical methods. Using two antibodies raised against human erythrocyte band 3 protein and two against human erythrocyte CA I and II isozymes, our study provides evidence that a protein related to band 3 is expressed in about 5% of cultured MDCK cells; these band 3-positive cells do not bind PNA and are not reactive for CAI or CAII. About 30% of the MDCK cells bind PNA, two-thirds of which are also CAII-positive. A majority (about 65%) of MDCK cells is not reactive for the three markers used; their density is increased after incubation with aldosterone. These data indicate (i) that the Cl(-)-HCO3- exchange of the MDCK cells could be related to human erythrocyte band 3, (ii) that the CA activity of the MDCK cell line bears antigenic identity with the erythrocyte CA II isozyme and (iii) that the latter is always co-localized with PNA binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Pituitary-dependent hormonal regulation of galaninergic neurons in the rat hypothalamus.

To investigate whether pituitary-dependent hormones may regulate galanin (GAL) content, synthesis and distribution in the hypothalamus, female hypophysectomized Wistar rats were treated for 2 weeks with subcutaneous injections of thyroxine (T4, 2 x 1 microgram), bovine growth hormone (GH, 2 x 125 micrograms), cortisol (C, 50 micrograms), subcutaneous implants of beta-estradiol (E2, 5-mm implant, dilution 1:1), or with the combinations [T4+GH], [T4+GH+C+E2] or [T4+GH+C+E2 + rat PRL, 2 x 125 micrograms] (doses/100 g BW/day). Concentrations of GAL in the hypothalamus were measured by radioimmunoassay (RIA) and GAL mRNA abundance was quantified by Northern blot (6 rats/group); 2 rats/group were used for immunohistochemistry. Hypophysectomy caused decreases of hypothalamic GAL peptide and mRNA concentrations (by 70 and 50%, respectively; p < 0.05 vs. intact rats). GAL immunoreactivity disappeared in the median eminence (ME), but increased in the neurohypophyseal magnocellular neurons of hypophysectomized rats. Substitution with T4, GH, [T4+GH], C or E2 had no significant effect on total hypothalamic GAL peptide and GAL mRNA concentrations. A treatment combining [T4+GH+C+E2] increased hypothalamic GAL (1.9 +/- 0.1 vs. 1.2 +/- 0.1 ng/mg protein in untreated hypophysectomized rats; p < 0.01) and GAL mRNA concentrations (127 +/- 19 vs. 59 +/- 2 densitometric units in untreated rats, p < 0.001). Addition of PRL to this combined treatment had no further effect. Treatment with T4, GH, [T4+GH] or E2 enhanced GAL labeling in the ME of hypophysectomized rats. The effect of estrogens was restricted to the GnRH-rich lateral regions of the ME. The combined treatment with [T4+GH+C+E2] restored the ME GAL immunoreactivity to levels observed in intact rats. In contrast, the increased GAL labeling observed in magnocellular neurons after hypophysectomy was not influenced by any hormonal treatment. In conclusion, hypophysectomy leads to marked reductions of hypothalamic GAL and GAL mRNA concentrations, and of GAL immunoreactivity in the ME. These reductions are prevented in part by a combined hormonal treatment associating T4, GH, C and E2, but not by any hormone given alone. This suggests specific pituitary hormone-dependent regulation of the hypophysiotropic GAL neurons. In contrast, the increased GAL labeling in magnocellular neurons of hypophysectomized rats persists despite hormonal treatment and likely represents a lesional effect on the neurohypophyseal GAL system.

Animals↗

PubMed1994

Expression of the endothelin-1 gene in the rat thyroid gland and changes in its peptide and mRNA levels in goiter formation and iodide-induced involution.

Endothelin-1 (ET-1) is a major vasoconstrictor peptide, first found in endothelial cells, and later in many other tissues, including the thyroid gland. We analysed the expression of the ET-1 gene in the rat thyroid gland and changes in ET-1 mRNA and peptide levels in goiter development and involution, two circumstances characterised by vascular changes. Thyroid hyperplasia was induced in adult Wistar rats by feeding a low iodine diet (LID) supplemented with 0.25% thiouracil for 10 days, and LID alone for 2 further days (H.12 group). Involution was induced by injecting 100 micrograms iodide and refeeding a normal diet during 6 h, 12 h, and 24 h (I.6h, I.12h, I.24h groups). Rats fed a normal iodine diet were used as controls. A specific 488 bp cDNA corresponding to the known sequence of pre-pro ET-1 was found by RT-PCR from RNA extracts in all thyroid experimental groups, as well as in lung and kidney which were used as positive controls. RP-HPLC analysis showed that ET-1 immunoreactivity eluted similarly as mature ET-1. During hyperplasia, ET-1 mRNA and peptide levels were increased 3.5- and 5-fold respectively. The relative volume of the vascular bed was more than doubled. During iodide-induced involution, the glandular ET-1 mRNA level remained elevated. The concentration of ET-1 peptide increased and was significantly greater at 12 h involution than in the H.12 group. At this time, the capillary reticulum reverted to individual capillaries and the vascular bed was significantly reduced. These data demonstrate that the ET-1 gene is expressed in the rat thyroid gland and that the ET-1 mRNA and peptide levels are increased during thyroid hyperplasia and remain elevated during a phase of rapid iodide-induced involution. These data suggest that changes in ET-1 production may play a role in control of thyroid gland trophic regulation and vascularity.

Animals↗

PubMed1994

Involvement of T cell immunity in the transient thyroid inflammation induced by iodide in goitrous BALB/C and nude mice.

To evaluate the involvement of T cell immunity in the thyroiditis induced in goitrous mice by iodide administration, we analyzed the immunological changes happening in the thyroid glands and lymph nodes during goiter involution in balb/c and athymic nude mice of similar background. In both balb/c and nude mice, goiter involution was characterized by thyroid cell necrosis and inflammation. In balb/c mice, the inflammatory infiltrate was made of numerous Ia+ cells. Their number was unchanged during goiter development, but was significantly increased after 2 days of involution and remained high after 8 days. CD4+ and CD8+ T cells were also observed, some of which were clearly activated since they expressed the receptor for Interleukin-2 (IL-2R). The numbers of CD4+, CD8+ and IL-2R+ T cells were increased during goiter as compared to control mice, and they reached a maximum at day 1 of involution. In nude mice, unexpectedly, CD4+ and CD8+ T cells were also found in the thyroid. Their numbers, as well as the number of Ia+ cells, were significantly increased at the onset of involution, but they remained systematically lower than in the corresponding groups of balb/c mice. Iodide treatment of goitrous mice also induced modifications of the lymph nodes draining the thyroid: enlargement of the paracortical T zone, presence of germinal centers in cortical follicles, and increase of the density of IL-2R+ cells. Mesenteric lymph nodes taken as controls were unchanged. Thus, three observations suggest the involvement of T cell immunity in iodine-induced thyroid inflammation: 1. Infiltration of Ia+, CD4+, CD8+ and IL-2R+ cells. 2. Signs of stimulation in thyroid lymph nodes, 3. Significant differences between balb/c and nude mice, in which the inflammatory reaction is weaker.

Animals↗

PubMed1993

Localization of a Band 3-related protein in the mitochondria-rich cells of amphibian skin epithelium.

Based on immunoblotting procedure, the isolated epithelium of amphibian skin was found to contain a 180 kDa protein which cross-reacts with a polyclonal antiserum raised against human erythrocyte Band 3. Immunoperoxidase and immunofluorescence staining techniques indicated that the Band 3-related protein was localized in the mitochondria-rich cells (MRC) of this epithelium, with characteristic apical labelling pattern. Our findings show that the putative apical anion exchanger of the MRC is immunologically related to the band 3 multigenic family, which catalyzes Cl(-)-HCO3- transmembranous exchange. It thus suggests a molecular basis for the role played by these cells in the transepithelial Cl- pathway and acid-base regulation.

Animals↗

PubMed1993

Effects of hypophysectomy on galaninergic neurons in the rat hypothalamus.

To understand better the relationship between hypothalamic galaninergic neurons and the pituitary gland, we studied the effects of hypophysectomy on hypothalamic galanin (GAL) content and distribution by radioimmunoassay and immunohistochemistry, and on GAL mRNA by Northern blot analysis. Three weeks after hypophysectomy, performed at 5 or 8 weeks of age, the hypothalamic concentrations of GAL and GAL mRNA were reduced by 30-50% in both male and female rats, compared to age- and sex-matched controls. Similar reverse-phase HPLC retention times of hypothalamic GAL were observed in intact and hypophysectomized rats. The reduction of hypothalamic GAL concentration following hypophysectomy was time-dependent, as peptide levels were unaffected one week after surgery. Immunohistochemistry showed regional differences in the effect of hypophysectomy on galaninergic neurons. In the hypophysiotropic hypothalamus, the scarce GAL immunoreactivity normally observed in the arcuate nuclei was no longer detectable in hypophysectomized rats, and the intense GAL immunoreactivity of the external zone of the median eminence progressively decreased and completely disappeared 3 and 6 weeks after hypophysectomy. In contrast, in the neurohypophyseal system, there was an increase of GAL labelling of the perikarya and emerging axons in the supraoptic and lateral-paraventricular nuclei, 1 and 3 weeks after hypophysectomy, that disappeared 6 weeks after hypophysectomy. An increase of GAL immunoreactivity was also observed in the internal zone of the median eminence 1 week but not 3 weeks after hypophysectomy. We conclude that hypophysectomy reduces the content of GAL and GAL mRNA in the rat hypothalamus. These changes are time-dependent and clearly detected after 3 weeks. The neurohypophyseal and hypophysiotropic galaninergic systems respond differently to hypophysectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Selenium deficiency aggravates the necrotizing effects of a high iodide dose in iodine deficient rats.

The effect of selenium deficiency associated with various iodide intake was investigated in rats in order to better understand its possible role in the etiopathogeny of myxedematous cretinism. Groups of rat pups were fed from birth a low selenium diet (Se-) and submitted to goitrogenic treatment (1% perchlorate in water) for one month. Some animals were refed iodide after perchlorate withdrawal. The gland morphology was analyzed in correlation with the glutathione peroxidase (GPX) activity and the thyroid hormone plasma levels. In all Se- rats, the GPX activity was strongly reduced as compared to selenium sufficient (Se+) animals (P < 0.01). Goitrous rats were hypothyroid whatever the selenium intake. After iodide refeeding, plasma T4 and T3 levels were increased by 160% in Se- rats and by respectively 330% and 580% in Se+ rats. The thyroid morphology was different according to the selenium intake: necrotic cells were about three times more numerous in Se- than in Se+ rats (P < 0.01) and the inflammatory reaction was increased. These experimental data demonstrate the detrimental role of selenium deficiency in one experimental case of thyroid disease. Such reduction of cell defences could contribute to the thyroid failure of African myxedematous cretins.

Animals↗

PubMed1993

High frequency of thymic ectopy in thyroids from autoimmune prone nonobese diabetic female mice.

BACKGROUND: The female nonobese diabetic (NOD) mouse, a well known experimental model to study autoimmune type 1 diabetes, also spontaneously develops thyroiditis. In this study, we report an abnormally high frequency of thymic ectopy in thyroids from NOD mice. EXPERIMENTAL DESIGN: Thyroids and thymuses from NOD mice and from control mice of different strains were processed for light and electron microscopy and for immunohistochemistry. RESULTS: Ectopic thymic tissue was observed in the thyroids of 80% of female NOD mice, whereas it was not found in control age-paired female mice from various other strains. The thymic tissue was present beneath the capsule as a large and unique fragment consisting of both a cortical dark one and a medullary light region. Thymic ectopies, as the thymus itself, contained thymocytes expressing both L3T4 and Lyt2 antigens, specific respectively for T helper and T suppressor/cytotoxic cells. Cortical and medullary epithelial cells were also easily identified by electron microscopy and by immunohistochemical staining using ER-TR4 and ER-TR5 monoclonal antibodies. CONCLUSIONS: Our observation could be an indication for a relation between abnormal thymus development and autoimmune disorder such as thyroiditis.

Animals↗

PubMed1992

Detection and identification of endothelin-1 immunoreactivity in rat and porcine thyroid follicular cells.

Endothelin-1 immunoreactivity (irET-1) was observed in rat and porcine thyroid glands. Using a radioimmunoassay for endothelin-1, the mean concentration in extracts of rat and porcine thyroid glands were 0.75 pg/mg +/- 0.03 (n = 4) and 1.5 pg/mg +/- 0.2 (n = 8) (mean +/- SE) respectively. Gel-filtration and reverse-phase HPLC showed that ir ET-1 eluted in a position identical to synthetic endothelin-1. In addition, immunohistochemical study showed that irET-1 is located within epithelial follicular cells. No immunostaining was seen in parafollicular C-cells nor in parathyroid.

Animals↗

PubMed1992

In vitro study of acute toxic effects of high iodide doses in human thyroid follicles.

The acute effects of increasing doses of sodium iodide were studied on human thyroid follicles isolated from normal paranodular tissue. After 24 h incubation in culture medium, follicles isolated from most thyroids maintained their capacity for 125I accumulation and organification and a normal cellular ultrastructure. 125I accumulation was significantly increased after addition of TSH, whereas 125I organification was not affected. In presence of TSH, numerous follicles had large empty-looking follicular lumina unlabeled on autoradiographies. Follicles incubated for 24 h in the presence of a low concentration (10(-7) M) of iodide retained their function and morphology. However, incubation with a high dose of iodide (10(-3) M) caused marked inhibition of 125I accumulation and organification reaching values similar to those obtained in presence of inhibitors of iodide trapping and organification. At high doses, iodide induced necrosis of thyroid epithelial cells: the percentage of necrotic cells was significantly increased with 10(-5) M and doubled with 10(-3) M as compared to values measured at 10(-7) M. Ultrastructural lesions such as apical blebbing, cytoplasmic fragments desquamation, endoplasmic reticulum vesiculation, and accumulation of lipofuscin in secondary lysosomes were also present. The necrotic effect and the ultrastructural alterations also occurred in the presence of TSH but were prevented by the addition of inhibitors of iodide trapping or organification. These results demonstrate a direct acute toxic effect of iodide in human thyroid cells. The nature of the ultrastructural alterations is in agreement with a mechanism of toxicity involving a free radical attack and lipid peroxidation as observed in other tissues.

Cytoplasm↗

PubMed1992

Iodine and goiter involution.

Iodine administration, although efficient in goiter treatment or prevention, is also responsible for adverse effects such as cell necrosis or thyroiditis. These two effects were reproduced in iodide-treated goitrous mice. Morphological observations strongly suggest that thyroid cell death results from an excessive production of free radicals, which initiates lipid peroxidation. This hypothesis is strengthened by the facts that the thyroidal concentration of malonic dialdehyde, a stable product of lipid peroxidation, is increased, and that necrosis is partially prevented by free radicals scavengers. Epithelial necrosis is associated to an inflammatory reaction. The infiltrate is mainly made of cells expressing class II molecules of major histocompatibility complex (macrophages and dendritic cells), but also of T lymphocytes. However, this inflammation, which varies among mouse strains, is transient and it is not amplified or maintained by administration of cytokines, IFN gamma or TNF alpha, known to induce class II expression on thyrocytes.

Animals↗

PubMed1990

Changes in cAMP-dependent and Ca2(+)-phospholipid-dependent protein kinase activities in suspension cultures of porcine thyroid cells.

The morphological and functional characteristics and the activities of cyclic AMP- (PKA I and PKA II) and calcium and phospholipid-dependent (PKC) protein kinases were studied in 2-day-old suspension cultures of porcine thyroid cells and were compared with those in freshly dissociated cells and intact glands. Thyroid cell morphology changed during the 2-day culture in the absence of specific regulators. This is characterized by a loss of cellular polarity, exo- and endocytotic vesicles and membranes of the rough endoplasmic reticulum, and an increase in the number of lysosomes, pseudomyelinic structures, lipidic inclusions and free ribosomes. Functional changes are characterized by a progressive decrease in protein iodination and its sensitivity to TSH stimulation. The total PKA activity in the cytosols of these cultures was slightly greater than that of freshly prepared tissue, due to the selective and significant accumulation of PKA I in cultured cells. In the particulate fraction the PKA activity was unchanged. PKC is the major kinase activity in porcine thyroids, and remains so in cultured cells. The slight drop in its activity in cytosols was offset by a significant increase in the particulate fraction, suggesting an intracellular redistribution of this kinase in cultured cells. The PKC activity is also partly activated in both the cytosol and particulate fraction, which results in an increased basal activity. The changes in PKA and PKC activities greatly modified the PKC/PKA ratios in the cytosols and the particulate fractions of cultured cells. These modifications could be partly responsible for the changes in sensitivity of cultured cells to the agents which control their activity.

Animals↗

PubMed1990

Correlated morphological and functional study of isolated rat thyroid follicles in suspension culture.

Rat thyroid follicles were isolated by collagenase digestion and cultured in suspension on agarose for 1-12 days with 0-0.1-1 mU/ml thyrotropin (TSH). After a 4 h exposure to Na125I they were processed for light and electron microscopy, autoradiography and biochemical analysis. Follicular 125I accumulation (A) and organification (PBI) were measured. Thyroglobulin (Tg) content of follicles and 125I-labelled amino acids in Tg were analyzed by high-performance liquid chromatography (HPLC). Without TSH, follicular lumina and cell polarity persisted. From day 3, the rough endoplasmic reticulum (RER) and ribosomes disappeared while autophagic vacuoles appeared: 125I accumulation and PBI were significantly reduced. From day 6, ultrastructural cell dedifferentiation occurred. At day 12, autoradiographic labelling was found over very few lumina; half of the 125I accumulated was still organified. With 1 mU TSH, follicles formed aggregates with narrow densely labelled lumina lined by tall cells. The RER was well developed up to day 12. 125I accumulation, PBI and iodothyronine (T3, T4) formation in Tg remained significantly higher than in follicles cultured without TSH, showing a transient decrease at days 6 and 9. Monoiodotyrosine/diiodotyrosine (MIT/DIT) and T3/T4 ratios in Tg were not modified, suggesting the persistence in the follicles of a significant iodine pool available for iodination. With 0.1 mU TSH, alterations of cell morphology and reduction of functional properties occurred later than without TSH. In the presence of TSH, morphological signs of new follicle formation were seen. These data demonstrate that closed follicles keep their follicular structure up to 12 days of culture, even without TSH. However, TSH is necessary to maintain iodine accumulation and organification.

Amino Acids↗