Chronic effect of TSH on human thyroid tissue in organ culture

The chronic effect of TSH on thyroidal cAMP concentrations and release of thyroid hormones was investigated using human thyroid tissue in organ culture. Normal human thyroid slices were placed in HAM's F-10 synthetic culture medium in Falcon organ tissue culture dishes, and incubated at 37 degrees in a humidified atmosphere of 5% CO2 in air. Medium was changed everyday and daily T3 or T4 release was determined using concentration of T3 or T4 in the medium. After incubation, slices were transferred to the medium containing 10 mM theophylline and incubated without TSH for an additional 30 min to determine thyroidal cAMP concentrations. Thyroidal cAMP concentrations in slices incubated with 10 mU/ml of TSH increased significantly at 2, 6, and 24 hr and even on the 6th day of incubation. Daily T3 release was significantly increased above control from the 3rd day and daily T4 release from the 4th day to the 11th day of incubation with 10 mU/ml of TSH. Histologically, almost all follicles were structurally maintained even on the 11th day of incubation. These results suggest that both thyroidal cAMP concentrations and release of thyroid hormones are stimulated chronically by TSH. This organ culture system is useful for investigating chronic effects of various materials on human thyroid tissue.     

The inhibitory effects of calmodulin antagonists on TSH-stimulated thyroid hormone release by mouse thyroid lobes

Calcium ions have been shown to play a mojor regulatory role in the release of various hormones from a wide variety of endocrine organs. More recently, in vitro evidence suggests that a calcium-binding protein, calmodulin, is also involved in the release of many hormones. So we examined the effects of several types of calmodulin antagonists on TSH-stimulated thyroid hormone release in vitro. Mouse thyroid lobes (one thyro-tracheal unit/tube) were incubated in Krebs-Ringer bicarbonate buffer at 37 degrees C for 4h. Free thyroxine (fT4) released in the incubation medium, thyroidal cAMP and calmodulin content were measured by RIA. TSH (5 mU/ml) and dibutyryl cAMP (DBC) (200 micrograms/ml) caused a 2-4 fold increase in thyroidal release of fT4. The stimulatory effects of TSH on fT4 release were significantly inhibited by trifluoprazine and prenylamine lactate at the concentration of 5 X 10(-5) M. More specific calmodulin antagonists, W-7 and W-13, were also shown to inhibit TSH stimulation of fT4 release at the concentration of 5 X 10(-5) M. In contrast, TSH stimulation of fT4 release was not depressed by non-specific antagonists, W-5 or W-12, at the same concentration as 5 X 10(-5) M. Further, W-13 also markedly inhibited DBC-stimulated fT4 release. Neither TSH nor PGI2 altered the thyroidal calmodulin content, dissociating with a marked increase in the cAMP concentration. These results suggest that calmodulin plays an important role in TSH-stimulated thyroid hormone release and further that this mechanism exists, at least in part, at the site subsequent to the generation of cAMP.     

Relation of structural polarity to responses of cyclic 3', 5'-adenosine monophosphate accumulation and thyroid hormone release in cultured human thyroid follicles

The relationship of structural polarity to functional activities was examined in cultured human thyroid follicles, which were isolated from the thyroid gland of patients with Graves' disease by collagenase treatment. Structural polarity was examined morphologically by electron microscopy, while the functional response to bovine TSH was examined by measuring intracellular cAMP accumulation and T3 release. In freshly isolated thyroid follicles, structural polarity was normal and TSH induced significant cAMP accumulation but no significant release of T3. After culture for 5 days the structural polarity of thyroid follicles became inverted in the absence of thyroid stimulators, but normal polarity was retained in the presence of TSH or dibutyryl cAMP [Bu)2 cAMP). The response to TSH of cAMP accumulation increased markedly after culture in either the presence or absence of TSH, suggesting that cAMP accumulation in response to TSH is not related to structural polarity. In contrast, thyroid follicles cultured without thyroid stimulators showed no significant T3 release in response to TSH, whereas those cultured with TSH or (Bu)2 cAMP showed significant T3 release in response to TSH. These data indicate that in cultured human thyroid follicles, the responses to TSH of cAMP accumulation and T3 release are not always correlated. Among many other explanations, the results were at least compatible with the idea that normal structural polarity is necessary for thyroid hormone release in response to TSH.     

Cyclic AMP release from perfused dog thyroid lobes as a sensitive indicator of TSH activation of the secretion process. Evidence for two types of thyroid secretion latency

It has been demonstrated in various types of thyroid tissue preparations that cyclic AMP (cAMP) released into the medium reflects the amount of cAMP in the cells. In the present study employing perfused dog thyroid lobes the dynamics of cAMP release were compared to those of thyroxine (T4) and triiodothyronine (T3) release. The experiments gave evidence that even the lowest concentrations of TSH which stimulate hormone release (in this study 1 microU/ml) also activate the cAMP system; the very high levels of cAMP obtained by stimulation with high concentrations of TSH (in this study 10,000 microU/ml) are not accompanied by corresponding high increases in hormone release. On the contrary the T4 and T3 release is lower than during stimulation with more moderate concentrations of TSH (100 microU/ml). Hence studies employing high concentrations of TSH and measurements of cAMP as indicator of activity of secretory processes should be interpreted very cautiously; the prolonged lag in thyroid hormone secretion observed after stimulation with low concentrations of TSH is accompanied by a corresponding lag in activation of the cAMP system. This pattern suggest that the duration of late secretory processes such as thyroglobulin pinocytosis and hydrolysis is independent of the degree of stimulation and not involved in the variations in secretion latency.     

Effects of somatostatin on human thyroid folliculi in vitro

Somatostatin (SRIF) inhibits calcitonin and T3-T4 secretion in thyroid. We have investigated the in vitro effect of SRIF on the basal and TSH induced [3H]thy incorporation, thyroglobulin (tgb) RNA and cAMP level in follicular cells, isolated from normal and adenomatous human thyroids. [3H]thy uptake has been evaluated as TCA-precipitable material in 2, 4, 8, 24 h incubated follicles and 24 h incubated adherent cells. Tgb RNA has been quantified with cytoplasmic dot blot hybridization and cAMP level with RIA method. SRIF reduces basal and TSH-induced [3H]thy in both suspension follicles and epithelial adherent cells. However it does not modify tgb RNA nor cAMP levels in incubated follicles. These data suggest a direct antiproliferative effect of SRIF on human thyroid.     

VIP and hormone secretion from thyroidal follicular and C-cells

The effect of vasoactive intestinal peptide (VIP) on the cAMP system of the thyroid and on the secretion of T4 and T3 from the follicular cells and calcitonin and somatostatin from the C-cells was studied in perfused dog thyroid lobes. Activation of the cAMP system was evaluated by measurements of the amount of cAMP released into the perfusion medium. T4, T3, calcitonin and somatostatin were measured by radioimmunoassays. 3 X 10(-6) M VIP induced increases in cAMP release and T4 and T3 secretion from the thyroid while there were no significant alterations in calcitonin and somatostatin release (n = 4). In experiments employing both of the two isolated thyroid lobes 100 microU/ml TSH gave considerably higher increases in T4 and T3 secretion than 10(-6) M VIP (n = 4). The effect of 10(-9) M VIP on T4 and T3 secretion was similar to that of 10(-6) M VIP (n = 4). 10(-10) M VIP induced a small but statistically significant increase in T4 and T3 secretion in two experiments while no effect was observed in two dogs. This high sensitivity of the follicular cells to VIP and the demonstration by others of VIP containing nerves in the thyroid suggest that VIP-ergic nerves may be involved in the regulation of thyroid hormone secretion.     

Increase of thyrotropin response to thyrotropin-releasing hormone (TRH) and TRH release in rats during pregnancy

Regulation of thyrotropin (TSH) release by thyrotropin releasing hormone (TRH) in the anterior pituitary gland (AP) of pregnant rats was studied. The pregnant (day 7, 14, and 21) and diestrous rats were decapitated. AP was divided into 2 halves, and then incubated with Locke's solution at 37 degrees C for 30 min following a preincubation. After replacing with media, APs were incubated with Locke's solution containing 0, or 10 nM TRH for 30 min. Both basal and TRH-stimulated media were collected at the end of incubation. Medial basal hypothalamus (MBH) was incubated with Locke's medium at 37 degrees C for 30 min. Concentrations of TSH in medium and plasma samples as well as the cyclic 3':5' adenosine monophosphate (cAMP) content in APs and the levels of TRH in MBH medium were measured by radioimmunoassay. The levels of plasma TSH were higher in pregnant rats of day 21 than in diestrous rats. The spontaneous release of TSH in vitro was unaltered by pregnancy. TRH increased the release of TSH by AP, which was higher in pregnant than in diestrous rats. Maternal serum concentration of total T3 was decreased during the pregnancy. The basal release of hypothalamic TRH in vitro was greater in late pregnant rats than in diestrous rats. After TRH stimulation, the increase of the content of pituitary cAMP was greater in late pregnant rats than in diestrus animals. These results suggest that the greater secretion of TSH in pregnant rats is in part due to an increase of spontaneous release of TRH by MBH and a decrease of plasma thyroid hormones. Moreover, the higher level of plasma TSH in rats during late pregnancy is associated with the greater response of pituitary cAMP and TSH to TRH.     

TSH stimulation of iodine organification in early foetal rat thyroid in vitro

Thyroid glands from 15 day-old rat foetuses were incubated in Eagle's medium containing Na 125I and supplemented, or not, with TSH for 4 or 24 hours. Electron microscopic radioautographic study shows silver grains mainly in follicular cavities only in the thyroids submitted to TSH during 24 hr. A functional differentiation must therefore take place in thyroid cells under TSH stimulation.     

The effect of propylthiouracil on thyroid-stimulating hormone-induced alterations in iodothyronine secretion from perfused dog thyroids

The aim of this study was to see whether the inhibitory effect of propylthiouracil on thyroidal secretion of 3,5,3'-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) could be reproduced in intensively stimulated thyroids, and to elucidate whether an increase in the fractional deiodination of thyroxine (T4) to T3 and rT3 during iodothyronine secretion might be responsible for the transient fall in the T4/T3 and T4/rT3 ratios in thyroid secretion seen in the early phase after stimulation of thyroid secretion. For this purpose T4, T3 and rT3 were measured in effluent from isolated dog thyroid lobes perfused in a non-recirculation system using a synthetic hormone free medium. 1 mmol/1 propylthiouracil induced a significant reduction in thyroid-stimulating hormone (TSH) stimulated T3 and rT3 release while the release of T4 was unaffected. This supports our previous conclusion that T4 is partially monodeiodinated to T3 and rT3 during thyroid secretion. Infusion of 1 mmol/l propylthiouracil for 30 min or 3 mmol/l propylthiouracil for 120 min did not abolish the transient fall in effluent T4/T3 and T4/rT3 induced by TSH stimulation. Thus, this phenomenon seems not to depend on intrathyroidal iodothyronine deiodinating processes.     

Effects of calcitonin and calcitonin gene-related peptide on 3H-thymidine incorporation into DNA of rat thyroid lobes in vitro.

The effects of a 4 h incubation of rat thyroid lobes, in the presence of calcitonin (CT) and calcitonin gene-related peptide (CGRP) on the incorporation of 3H-thymidine into DNA, were investigated. In other groups the thyroid lobes were incubated during exposure to CT and thyrotropin (TSH), and to CGRP together with TSH. All concentrations of CT (10(-6)-10(-8) M) revealed a tendency towards lowering 3H-thymidine uptake, but the effect was not statistically significant. The influence of CGRP was dose-dependent; the lowest concentration of CGRP (10(-9) M) significantly enhanced DNA synthesis in the incubated rat thyroids; an intermediate dose of the peptide (10(-8) M) had no effect, while the highest concentration of CGRP (10(-7) M) decreased 3H-thymidine incorporation. Calcitonin (10(-7) M), as well as CGRP (10(-8) M), suppressed the stimulatory effect of TSH on 3H-thymidine incorporation.     

Dynamics of hormone release from the perfused canine thyroid during cyclic AMP infusion.

In order to try to characterize the sequence of processes leading to hormone secretion from the stimulated thyroid, the effect of cyclic 3'5' adenosine monophosphate (cAMP) and related compounds were examined in 15 two-sided perfusions of canine thyroids isolated in situ. T4 and T3 concentrations in the effluent were measured radioimmunologically. cAMP 5 mM and TSH 100 muU per ml induced the same pattern of hormone release from the thyroid. After a latency period of 15--25 minutes a steep increase occurred in both T4 and T3 release. During the initial part of the stimulation the rise in T4 relase was somewhat slower than that of T3 release. The prolonged latency period before response earlier recorded in the same preparation during infusions of low concentrations of TSH was not observed during infusions of decreasing concentrations of cAMP (1, 0.8, 0.5 and 0.2 mM) or theophylline (5 and 1 mM). Either there was no response or the latency period was of the same length as that observed after a strong stimulus. These findings suggest that the latency period can be divided in two parts: () a variable, dose dependent satency period confined to the early part of the process sequence leading to secretion--i.e. before cAMP exerts its effect, and 2) an obligatory latency period related to the processes taking place after the formation of pseudopods. The duration of these late processes seems to be independent of the degree of stimulation.     

Iodotyrosine deiodination in the normal and acutely TSH-stimulated thyroid.

The deiodination of L-MIT-125I was measured in rat thyroid homogenates and slices before and after acute TSH stimulation. Slices and homogenates were incubated with identical concentrations of tissue and substrate in the presence and absence of NADPH. 1 USP unit TSH added in vitro to thyroid slices failed to stimulate deiodination; a single in vivo ip injection of 3 USP units TSH was also unable to raise deiodinating activity. In contrast to TSH, NADPH added to homogenates and slices enhanced deiodination significantly. However, several arguments, including a review of the literature, strongly militate against the hypothesis of an increased intracellular concentration of the coenzyme NADPH being the prerequisite to enhanced deiodination. The results suggest that deiodinase activity in acutely stimulated thyroids is not limited by the intracellular concentration of the enzyme itself nor by the availability of co-enzyme. Therefore, the increased iodide release induced by acute TSH stimulation is a mere consequence of the enhanced thyroglobulin proteolysis and does not require higher enzyme concentration. It will be shown subsequently that a different conclusion must be drawn in experiments with chronic TSH stimulation.     

The morphologic and physiologic behaviour of thyroid lobes from newborn rats during short periods of incubation in vitro

Under light and electron microscopic examination, the morphology of thyroid lobes obtained from newborn rats remained essentially normal during periods of incubation in vitro lasting as long as several hours. In response to the presence of TSH in the incubation medium, follicular cells of the lobes extended long microvilli into the follicular lumen and ingested large droplets of colloid. The ability of the lobes to carry out essential steps in the synthesis and release of thyroid hormone during incubation was demonstrated by radiochromatographic analyses. Stimulation with TSH increased the amount of iodide taken up from the medium by the lobes.     

Influence of cAMP and calcium on epinephrine-mediated hepatic glycogenolysis in rainbow trout,Oncorhynchus mykiss

1. The role of cAMP and of calcium in mediating epinephrine-stimulated glycogenolysis was studied by incubating rainbow trout liver in vitro.2. Epinephrine significantly stimulates glucose release from liver pieces incubated in either calciumcontaining or calcium-free medium. However, the development of the glycogenolytic profile occurred more rapidly in the presence of calcium.3. The β-antagonist, propranolol, inhibited epinephrine-stimulated glucose release from liver pieces incubated in either calcium-containing or calcium-free medium.4. Calcium ionophore, A3187, stimulated glucose release from liver pieces incubated in calciumcontaining medium. Verapamil, a putative calcium channel blocker, had no effect on A23187-stimulated glycogenolysis. However, verapamil completely inhibited epinephrine-stimulated glycogenolysis.5. Dibutyryl cAMP and IBMX, singly or together, stimulated glucose release from liver pieces. cAMP-mediated glycogenolysis was more pronounced in liver pieces incubated in calcium-containing medium.6. These results indicate that epinephrine-stimulated hepatic glycogenolysis in rainbow trout proceeds through the activation of β-adrenergic receptors and that both cAMP and calcium are involved in the post-receptor signal transduction process.     

A BIOCHEMICAL AND RADIOAUTOGRAPHIC ANALYSIS OF PROTEIN SECRETION BY THYROID LOBES INCUBATED IN VITRO

In this study we analyzed several aspects of protein secretion by thyroid follicular cells. The study was carried out on intact thyroid lobes obtained from newborn rats and incubated in vitro. The fate of leucine-³H incorporated into protein within follicular cells of untreated and thyrotropic hormone (TSH)-treated lobes was traced by quantitative electron microscope radioautography. Our findings indicate that protein synthesized by the rough-surfaced endoplasmic reticulum during a pulse exposure to leucine-³H is released relatively slowly by this organelle. Approximately 1 hr after onset of the pulse, a peak of radioactive protein appears in the Golgi region. The significance of this peak is not clear. Newly synthesized secretory protein passes through the apex of follicular cells without being concentrated or temporarily stored there in the form of large secretory droplets. Passage probably takes place via small vesicles which are intermingled among diverse small vesicles at the apex of the cells as well as in the Golgi region. Exposure of the lobes to TSH in the incubation medium for 45 or 90 min does not stimulate incorporation of leucine-³H into protein. Acute stimulation with TSH does, however, modify the movement of secretory protein within the exocrine secretory apparatus of the follicular cell. It accelerates the arrival of the protein at the apex of follicular cells, and it accelerates the release of the protein into the follicular lumen.     

Lack of effect of propylthiouracil and methylmercaptoimidazole on thyroglobulin biosynthesis

Experiments were performed both in vivo and in vitro to test a previous proposal that part of the antithyroid action of the thioureylene drugs, propylthiouracil (PTU) and methylmercaptoimidazole, can be attributed to inhibition of thyroglobulin (Tg) biosynthesis. Rat thyroid lobes were incubated in leucine-free Eagle's medium containing bovine thyroid-stimulating hormone and 0, 0.1-0.2, or 1 mM drug. After a 30-min preincubation, 5 mu Ci of [14C]leucine were added and the incubation was continued for 4 hr. The soluble fraction was analyzed by sucrose density gradient centrifugation, and the fractions corresponding to the 19S Tg peak were pooled and assayed for 14C. No inhibition of 14C incorporation into 19S Tg was observed, even in thyroid lobes incubated in the presence of 1 mM methylmercaptoimidazole or 2 mM PTU. At the same time, 14C incorporation into 19S Tg was completely inhibited when lobes were incubated in the presence of 0.1 mM puromycin. In vivo, rats received an injection of PTU (1 mumol/100 g body wt), followed 60 min later by an injection of 25 mu Ci of [14C]leucine. Blood samples and thyroids were taken 5 hr after the [14C]leucine injection. Serum thyroid-stimulating hormone was not significantly affected by the PTU injection. The thyroid-soluble fraction was analyzed by sucrose density gradient centrifugation. No significant differences between saline and PTU-injected groups were observed in [14C]leucine incorporation into 19S Tg. We conclude from both our in vitro and our in vivo studies that PTU and methylmercaptoimidazole have no inhibitory effect on thyroglobulin synthesis in rat thyroids and that such inhibition does not play a significant role in the antithyroid action of these drugs.     

Interaction of VIPergic and cholinergic receptors in human thyroid cell

The thyroid tissue is innervated by cholinergic and VIPergic nerves. The present study investigated the possible interactions of cholinergic agents with VIP-induced cAMP accumulation and thyroid hormone release in vitro. Carbamylcholine (Cch), acting through the muscarinic receptor increases cellular cGMP content in cultured human thyroid cells incubated with a phosphodiesterase inhibitor. Cch (10 microM) inhibits cellular cAMP accumulation and thyroxine (T4) release induced by vasoactive intestinal peptide (VIP), with or without a phosphodiesterase inhibitor. Cch (10 microM) inhibits 8-bromo-cAMP-induced T4 release from human thyroid slices. 8-Bromo-cGMP inhibits VIP-induced T4 release from human thyroid slices, only in cells incubated without the phosphodiesterase inhibitor. The results indicate that interactions between VIPergic and cholinergic receptors may be of importance in human thyroid cell.     

"In vitro" study on release of cyclic AMP and thyroid hormone in autonomously functioning thyroid nodules

The TSH effect on slice and the incubation medium cyclic AMP levels and T3 and T4 released from 8 autonomously functioning thyroid nodules (AFTN) and their respective perinodular (PN) tissues were examined. The thyroid slices were incubated in Eagle's Medium containing TSH (5 to 100 mU/ml) for 60 min and 300 min for tissue cyclic AMP generation and for cyclic AMP, T3 and T4 release, respectively. Basal cyclic AMP levels were not different either in AFTN and in PN slices or into the incubation medium. In both tissues TSH produced a similar cyclic AMP generation. In contrast, cyclic AMP released into the incubation medium was significantly higher in AFTN than in PN tissues, after TSH stimulation. Basal T3 values and TSH-stimulated T3 release in AFTN were not different from PN tissue. However, basal T4 levels were significantly higher in AFTN than in PN tissue as well as T4 released in response to TSH. In addition, T3/T4 ratio was lower in AFTN than in PN tissues. The cyclic AMP released into the incubation medium correlated with both T3 and T4 release in PN tissue but in the AFTN tissue no correlations were found. These findings suggest that the adenylate cyclase-cyclic AMP system is more sensitive to TSH-stimulation in AFTN when compared with PN tissue and that AFTN tissue has a preferential T4 secretion.     

Direct effects of trypan blue on thyroid secretion.

Trypan blue directly inhibited in vitro thyroid secretion (butanol soluble 125I release to the media) induced by both thyroid stimulating hormone (TSH) and dibutyryl cAMP. Intracellular colloid droplet counts were also decreased. Inhibition was directly proportional to dye concentration and could be overcome by supramaximal TSH and dibutyryl cAMP. Inhibition could be observed as early as 20 min of incubation, was not increased by preincubation, and could even be demonstrated after TSH in vivo. Trypan blue, in vivo, produced similar inhibition of thyroid secretion. Incubation of 125I-thyroglobulin with lysosomal enzymes revealed inhibition with much lower concentrations of dye. Inhibition of lysosomal enzyme(s) would not appear to explain the marked decreases in colloid droplets, and this may represent two separate effects of trypan blue on thyroid secretion.     

Prevention by nicotinamide of desensitization to thyrotropin stimulation in cultured human thyroid cells

The presence of 50 mM nicotinamide together with 100 milliunits/ml of TSH in the incubation medium prevented the decline in human thyroid cell cAMP from maximum, stimulated levels (15-30 min) that occurs when the cells are exposed to TSH alone. Nicotinamide in the absence of TSH did not increase thyroid cell cAMP content. TSH desensitization, and its prevention by nicotinamide, occurred in the presence or absence of 3-isobutyl-methylxanthine. 1-Methyl nicotinamide and N'-methyl nicotinamide similarly prevented TSH desensitization. Recovery from TSH desensitization was prolonged and incomplete after 72 h. The presence of 50 mM nicotinamide hastened recovery from desensitization. Desensitization of the cAMP response to 10(6) M prostaglandin E1 and 1 mM adenosine was unaffected by nicotinamide. Other inhibitors of poly(ADP-ribose) polymerase activity, 5-bromouridine, 5-bromo-2'-deoxyuridine, and thymidine (all at 50 mM) completely or partially prevented TSH desensitization. Pyridoxine (50 mM) similarly prevented this phenomenon. As with dog thyroid cells, 10(-4) M cycloheximide blocked TSH desensitization. The combination of 10(-4) M cycloheximide and 50 mM nicotinamide had a synergistic effect in augmenting the thyroid cell cAMP response to TSH stimulation.