The mechanism of damping of the serum thyroxine and triidothyronine levels caused by increasing thyrotropin dosage in mice.

The time course of the effect of bovine TSH (bTSH) on serum concentrations of thyroxine (T4) and triiodothyronine (T3) was measured in the normal mouse. The basal, unstimulated levels were 3.2+/-1.1 mug/100 ml T4 and 104+/-25 ng/100 ml T3 (mean+/-SD). With doses of bTSH from 0.5 to 100 mU the peak levels of the thyroid hormones were only 2.6 and 1.8 times the basal level for T4 and T3, respectively. With increasing doses of bTSH there was a proportional prolongation of the increased serum levels of thyroid hormones, i.e., about 2 h for 0.5mU to 12 h for 100 mU TSH. The integrated response with time was linearly related to the log dose. This would suggest a control mechanism which prevents excessive concentration of thyroid hormones in the serum. This pattern of response to TSH differs somewhat from that obtained by following radioiodine release in the McKenzie type bio-assay. To avoid the problems of changing blood concentrations of thyroid hormones and TSH, the release of T4 and T3 from the mouse thyroid was measured in vitro. The secretion increased with bTSH concentrations in the range of 0.02-0.8 mU/ml for T4 and 0.02-0.4 mU/ml for T3. The maximal response was 8.8+/-0.5 ng T4/3h/thyroid and 3.6+/-0.3 ng T3/3h/thyroid as against the basal secretion of 2.4+/-0.2ng T4 and 0.8+/-0.1 ng T3 (mean+/-SEM). Further in crease in bTSH concentration was associated with a decreased rate of thyroid hormone release. Thyroidal cAMP accumulation was enhanced with increasing bTSH concentration, even when there was a decrease in secretion. The dichotomy in the dose-response pattern between the two parameters indicated that the effect of high TSH concentrations on the release was induced at a step beyond cAMP accumulation. This was corroborated by the similar pattern of release induced by increasing concentration of DBcAMP.These findings indicate the existence of an intrathyroidal autoregulatory mechanism which prevents excess increase of thyroid hormone levels in the blood.     

Forskolin-induced elevation of cyclic AMP does not cause exocytosis and endocytosis in rat thyroid follicle cells in vivo

Using a newly developed infusion technique, the in vivo effects of forskolin and dibuturyl cyclic AMP (dbcAMP) on exocytosis and endocytosis in thyroid follicle cells were studied in thyroxine-treated rats and mice. Reactants were selectively infused via the superior thyroid artery to one thyroid lobe. The contralateral lobe served as a control. In the rat, a supramaximal i.v. dose of thyrotropin (TSH, 500 mU) induced a slight increase in thyroidal tissue levels of cyclic adenosine monophosphate (cAMP) while TSH 50 mU i.v. had no effect on cAMP levels. On the other hand both doses of TSH stimulated exocytosis, signified by a decrease in the number of exocytotic vesicles and endocytosis, signified by the appearance of pseudopods and colloid droplets. Selective thyroid infusion of dbcAMP (5 mM) or forskolin (25 microM), which induced a 10-fold increase in thyroid cAMP levels, did not induce any morphological sign of exocytosis or endocytosis in the follicle cells. The morphological response to TSH given i.v. was quantitatively unaltered by simultaneous infusion of forskolin. In contrast to the findings in rats, infusion of forskolin and dbcAMP in mice induced endocytosis. In conclusion, our findings in the mouse are in agreement with earlier studies in this and other species, indicating that cAMP mediates the effects of TSH on endocytosis and probably also on exocytosis. In contrast, our observations in the rat thyroid in vivo lead to the conclusion that cAMP is not the main intracellular mediator of exocytosis and endocytosis in this species. This conclusion is at variance with previous reports, mostly from in vitro studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Triiodothyronine and 3',5'-cyclic AMP secretion by cultured human thyroid cells in response to thyrotropin and thyroid-stimulating immunoglobulin

We have established a relatively simple and sensitive system for measuring T3 as well as cAMP secretion using cryopreserved human thyroid cells in culture. We defined optimal culture conditions and characterized the system. T3 secretion from human thyrocytes (only 1 x 10(5) cells/well) could be stimulated in a time- and dose-dependent fashion by both TSH (doses as low as 10 mU/l) and thyroid-stimulating immunoglobulin to levels 5- to 10-fold above baseline. The response to the thyroid stimulating agents was preserved for at least 3 weeks. Experiments with inhibitors of iodothyronine synthesis (propylthiouracil and methimazole) indicated that the bulk of the TSH-stimulated T3 secretion measured apparently derives from de novo iodothyronine biosynthesis rather than preformed T3. We utilized the system to investigate some aspects in the regulation of human thyrocyte T3 and cAMP secretion. Maximum stimulation of the thyroid hormone was achieved at TSH doses capable of evoking a further rise in levels of cAMP. A rise in cAMP accumulation was observed as early as 15 min following exposure to TSH, whereas it took 1-4 days to detect a significant increase in T3 secretion. Within 6 h of incubation, the bulk of TSH-stimulated intracellular cAMP was found released into the medium. 1-methyl-3-isobutylxanthine (MIX) caused a dose-related decrease (beyond 0.1 mmol/l MIX) in TSH-stimulated T3 secretion which contrasted with a concomitant expected increase in cAMP accumulation. Hence, as also observed in adrenal and testicular tissue, xanthines at high concentration seem to exhibit a dual action: potentiation of cAMP accumulation by inhibiting phosphodiesterase activity and a concomitant reduction of hormone formation.     

Inhibition of the response of mouse thyroid to tryrotropin induced by chronic triiodothyronine treatment.

Administration of 1 mU bovine TSH iv to mice resulted, within 1 hour, in the increase of the serum T4 level from 32 +/- 1.4 ng/ml to 53 +/- 2.6 ng/ml (Mean +/- SE, n = 24). Treatment with 1 mug triiodothyronine (T3) per day, for 10 days, abolished the responsiveness of the thyroid to TSH, as measured by thyroxine (T4) release. Thyroidal response to TSH was measured also in vitro. The basal hormonal release was 4.66 +/- 0.55 ng T4 and 0.98 +/- 0.15 ng T3 per thyroid per 3 h (n = 30). In the presence of bovine TSH (0.2 mU/ml) the hormonal secretion increased 3-fold for T4 and 2.5-fold for T3. Thyroids from mice pretreated with T3 for 10 days showed almost no response to TSH. Partial refractoriness to TSH was already significant 5 days after T3 pretreatment. Responsiveness to TSH was restored 3 days after T3 withdrawal or after 3 daily injections of 10 mU bovine TSH, concomitant with the last 3 days of T3 pretreatment. These results indicated that the prolonged absence of an adequate level of trophic hormone may be the cause of thyroidal unresponsiveness to acute TSH treatment. With 20 mU of TSH, cAMP levels rose from 4 +/- 0.5 picomoles to 80 +/- 9.3 picomoles per thyroid (n = 6). In mice subjected to 10 days of T3 pretreatment the response was markedly reduced: 20 +/- 3 picomoles/ thyroid. Thyroids of the T3-treated mice responded normally to 1 mM DBcAMP in vitro. From these results it was concluded that the impaired responsiveness of the thyroids to TSH occurs at a step prior to cAMP accumulation.     

Effects of drugs that modify brain biogenic amine concentrations on thyroid activation induced by exposure to cold.

L-Dihydroxphenylalanine (L-DOPA) significantly inhibited intrathyroidal colloid droplet formation induced by exposure to cold in the rat. Diethyldithiocarbamate (DDC) also inhibited colloid droplet formation in response to cold. The combined administration of L-DOPA and DDC produced an additive inhibition of the thyroidal endocytotic response to exposure to cold. Pretreatment with chlorpromazine (CPZ) ameliorated the inhibitory effect of L-DOPA. DL-alpha-methyl-p-tyrosine (alpha-MT) also signficantly depressed the thyroidal response. Inhibition of colloid droplet formation induced by alpha-MT was not altered by the administration of DL-dihydroxyphenylserine (DL-DOPS). On the other hand, treatment of the alpha-MT-treated rats with L-DOPA to normalize dopamine synthesis resulted in a dramatic recovery from the inhibition. Blockade of serotonin biosynthesis with p-chlorophenylalanine (p-CPA) failed to produce a significant inhibition of colloid droplet formation. However, 5-hydroxytryptophan (5-HTP) markedly inhibited the thyroidal response to cold. Brocresine phosphate (BP) was another inhibitor of the thyroidal endocytotic response to exposure to cold. Oxotremorine also markedly depressed the thyroidal response to cold. Since these drugs did not interfere with pituitary thyroid responsiveness to exogenous thyrotropin-releasing hormone (TRH), it seems that the throidal endocytotic response to exposure to cold as a reflection of TSH secretion was directly influenced by alterations of brain biogenic amine concentrations or turnover rates.     

The biphasic stimulatory and inhibitory effects of concanavalin A on thyroid activation induced by thyrotropin.

Concanavalin A (Con A) was tested for its ability to affect thyroid activation induced by TSH in mouse thyroid lobes. Pretreatment of thyroid lobes with Con A at concentrations from 1.55--400 microgram/ml was found to have biphasic stimulatory and inhibitory effects of the TSH-induced accumulation of cAMP and formation of colloid droplets. Low concentrations of Con A potentiated TSH activation of thyroidal formation of cAMP and endocytosis. In contrast, higher concentrations of Con A markedly inhibited these TSH effects. The inhibitory effects observed after preincubation with Con A were abolished by the addition of alpha-methyl-D-glucoside to the incubation medium. A high concentration of Con A also inhibited cAMP formation induced either by prostaglandin E2 or the long-acting thyroid stimulator. However, the basal and TSH-stimulated glucose oxidation in mouse thyroid lobes was not depressed by a high concentration of Con A. Uptake of 125 I-labeled Con A by thyroid tissues increased with time up to 1 h and was directly proportional to tissue weight. These findings suggest that the specific interaction between Con A and its receptors may lead to conformational changes in the structure of the membranes of the thyroid follicular cells which facilitate TSH-induced thyroid hormone secretion via the adenylate cyclase-cAMP system.     

Characterization of T3 immunoreactivity release from thyroid gland in vitro a reflection of colloid droplet formation

T3 immunoreactivity release from the thyroid gland in vitro was shown to be increased by TSH. In the present study, we sought to determine whether the T3 immunoreactivity release is an indicator of thyroid hormone secretion or due to hormone synthesis. When thyroid glands from mice were incubated with TSH, T3 immunoreactivity release was increased in parallel with intracellular colloid droplet formation in a dose related manner. When colchicine, a known inhibitor of colloid droplet formation, was added, both T3 immunoreactivity release and colloid droplet formation were inhibited, whereas MMI, an inhibitor of hormone synthesis, failed to influence both aspects. Thus T3 immunoreactivity release as a reflection of colloid droplet formation was demonstrated. The analysis of the released immunoreactivity by Sephadex column and subsequent radioimmunoassay suggested that the T3 immunoreactivity was, to a considerable extent, due to macromolecule instead of T3 itself. The effect of I- or Li+ to inhibit thyroid hormone secretion was shown to be on the step prior to, but not subsequent to, colloid droplet formation.     

In vitro studies on the nature of vinblastine inhibition of thyroid secretion.

The effects of vinblastine on thyroid secretion have been studied in mouse thyroid glands in vitro. Inhibition of the secretory response to TSH was observed within the first 30 min of exposure to vinblastine and was largely complete after 1 h. Inhibition of colloid droplet formation and hormonal radioiodine release was comparable for both TSH and cyclic AMP stimulation. No reversal of inhibition was seen when thyroids exposed to vinblastine were subsequently washed 2 or 4 h in vinblastine free mediu, nor could previous vinblastine inhibition be overcome by high levels of TSH or dibutyryl cyclic AMP. Ultrastructural effects of vinblastine (1 X 10(-5)M) were studied under conditions similar to those used to demonstrate inhibition of secretion. Microtubules present in thyroids incubated in control medium were reduced after 30 min and almost completely abolished after 1 or 2 h exposure to vinblastine. No other reproducible structural changes were seen. A higher concentration of vinblastine (5 X 10(-5)M), however, produced lysosomal clumping and the formation of numerous vinblastine crystalloids. Uptake and washout of [3H]-vinblastine were studied with both phases showing fast and slow components. The initial uptake phase (T1/2 approximately 12 min) correlates well with the disappearance of microtubules and the inhibition of thyroid secretion. A prolonged washout component (T1/2 = 10-18 h) may account for the irreversibility of vinblastine inhibition of in vitro thyroid secretion. It is concluded that inhibition of thyroid secretion produced by vinblastine is consistent with its actions on thyroidal microtubules.     

Levothyroxine dose requirements for thyrotropin suppression in the treatment of differentiated thyroid cancer.

We have compared the dose of levothyroxine (L-T4) required to suppress serum TSH to given levels in two clinical groups: 1) 44 patients with thyroid cancer whose thyroid glands had been ablated by surgical thyroidectomy and 131I treatment, and 2) 113 patients with thyroidal failure due either to spontaneous primary hypothyroidism (31 patients) or after 131I treatment for Graves' hyperthyroidism (82 patients). The dose of L-T4 needed to attain serum TSH levels in the euthyroid range (0.5-6.2 microU/mL) was significantly greater (P less than 0.01) in patients with thyroid cancer (2.11 micrograms/kg.day) than in the patients with primary hypothyroidism associated with nonmalignant disease (1.63 micrograms/kg.day). Similarly, patients with thyroid cancer required a higher dose of L-T4 to suppress serum TSH to a given subnormal level. These findings suggest that the secretion of hormone from residual thyroid tissue in patients who have not been subjected to near-total thyroid ablation contributes substantially to the circulating levels of serum T4 and T3. We, therefore, infer that residual thyroidal secretion in the patients with hypothyroidism due to benign causes is relatively independent of TSH stimulation. Further subdivision of patients with benign hypothyroidism revealed that patients with Graves' who developed hypothyroidism after 131I treatment showed a lower mean dose requirement than patients with spontaneous hypothyroidism. This raises the possibility that continued secretion of thyroid-stimulating immunoglobulin in such patients might account for the lower dose requirement in the combined group with hypothyroidism. Our studies also have allowed us to make serial observations in 4 patients with thyroid cancer who exhibited elevated levels of serum thyroglobulin. In this limited series, maximal suppression of serum thyroglobulin was produced by doses of L-T4, which reduced circulating TSH to 0.4 mU/L.     

Progressive recruitment of follicular cells with graded secretory responsiveness during stimulation of the thyroid gland by thyrotropin

One of the earliest responses of the thyroid cells to TSH is macropinocytosis with formation of intracellular colloid droplets. We demonstrate here that increasing stimulation with TSH not only elicits a highly individual macropinocytotic response among different follicular cells but that the fraction of TSH-responsive cells is also a function of the TSH dose. After pretreatment with T4, mice and rats were injected ip with bovine TSH and killed 2 h later. The macropinocytotic response to TSH was evaluated on periodic acid-Schiff-stained 3-microns sections of the thyroids in terms of droplet number per 25 follicles and, in addition, by assessing recruitment, i.e. percentage of droplet-containing cells. Both variables increased with increasing TSH stimulation until they reached a plateau at about 9 mU TSH in mice and at about 300 mU TSH in rats: the percentage of droplet-containing cells gradually increased in mice from 2% (no TSH) to 67% (9 mU TSH) and in rats from 11% (no TSH) to 54% (300 mU TSH). Overall pinocytotic response as well as thyrocyte recruitment could be modified by extra- and intrathyroidal factors: for example, pretreatment of the mice with an iodine-deficient diet increased the maximal percentage of droplet containing cells to nearly 90%. Obviously, two separate components of the macropinocytotic response of the thyroid gland to TSH can be distinguished: the first is the gradually increasing fraction of droplet-containing cells, the second is the well known increase of the number of colloid droplets in each TSH-responsive cell with progressive TSH stimulation. Recruitment of thyrocytes with a gradually increasing natural threshold to a hormonal stimulus appears to be a fundamental mechanism in the thyroid gland and possibly in other organs.     

The role of the pentose phosphate shunt in thyrotropin-induced thyroid hormone secretion: in vivo and vitro studies with 6-aminonicotinamide in mouse thyroids.

The possible role of the pentose phosphate shunt in thyroid hormone secretion was investigated in vivo and in vitro with mouse thyroid glands. Thyroidal endocytosis in response to TSH, a step of thyroid hormone secretion, was evaluated for its dependency upon the pentose phosphate shunt by using 6-aminonicotinamide (6-AN), an antimetabolite in the synthesis of pyridine nucleotides. Formation of 14CO2 from glucose labeled either in the C-1 or C-6 position was studied to estimate the pentose phosphate shunt activity. A dose of 6-AN markedly reduced oxidation of [1-14C]glucose but did not affect that of [6-14C]glucose induced by TSH. Concomitantly there was a marked decrease in thyroidal endocytotic response to TSH. These inhibitions by 6-AN were completely abolished by the pretreatment with nicotinamide. Methylene blue, which oxidizes NADPH and thus stimulates activity of the pentose shunt, significantly depressed thyroidal endocytosis in response to TSH in vitro. These inhibitions of colloid droplet formation by 6-AN or methylene blue were not manifested against dibutyryl cyclic AMP stimulation. Furthermore, a dose of 6-AN, which seems to inhibit only the pentose phosphate shunt, markedly depressed TSH-induced formation of cyclic amp. These findings suggest that the pentose phosphate shunt might play an important role in triggering TSH stimulation of thyroid hormone secretion by supplying NADPH, and further, that NADPH dependency in thyroid hormone secretion is at a site prior to the generation of cyclic AMP.     

Plasma thyrotropin bioactivity in Down's syndrome children with subclinical hypothyroidism

OBJECTIVE: Subclinical hypothyroidism occurs in a number of children with Down's syndrome (DS). The reason for the mildly elevated plasma thyrotropin (TSH) concentrations is not known. The present study investigated whether decreased TSH bioactivity plays a role in this phenomenon. DESIGN: A retrospective study of plasma specimens from DS children with mildly elevated plasma TSH concentrations and thyroid hormone levels within the reference range, using a TSH receptor-adenylate cyclase mediated bioassay. METHODS: Strain JP26 Chinese hamster ovary (CHO) cells, stable transfected with the human TSH receptor, were incubated with unfractionated plasma (1/10 diluted in hypotonic incubation medium) of 10 DS children with subclinical hypothyroidism and nine euthyroid children with insulin-dependent diabetes mellitus as controls. cAMP released in the incubation medium was measured by RIA. Mock-transfected CHO cells were used to correct for non-specific CHO response. WHO Second International Reference Preparation of human TSH was dissolved and diluted in pooled normal human plasma and simultaneously bioassayed to match patient and control results. RESULTS: Plasma TSH levels were slightly increased in DS (mean +/- S.D., 6.5+/-1.3 mU/l, reference range 0.4-4.0 mU/l). Plasma TSH levels for controls (1.3+/-0.4 mU/l) were within the reference range. Plasma thyroid hormone levels in patients and controls were normal, plasma TSH binding inhibitory immunoglobulin and thyroid peroxidase antibodies were negative. cAMP levels (corrected for non-specific CHO response) in DS patients (18.4+/-3.9 pmol/well) and in controls (14.3+/-1.3 pmol/well) did not significantly differ from cAMP levels generated by patient-TSH equivalent TSH standards (16.3+/-0.9 pmol/well). CONCLUSIONS: The present results demonstrate normal TSH bioactivity in plasma of DS children, indicating that subclinical hypothyroidism in these patients is of primary (thyroidal) origin.     

Decreased thyroidal response to thyrotropin in diabetic mice

The effect of diabetes mellitus on the synthesis and secretion of thyroid hormone ws investigated in mice with streptozotocin-induced diabetes. Thyroid glands were labeled in vivo with 131I for 2 h. In control animals, TSH stimulated the synthesis of PB127I and 131I-labeled iodothyronines and simultaneously decreased the proportion of 131I-. These effects of TSH were not observed in diabetic animals but were demonstrable in diabetic animals treated with insulin. For studies of hormone secretion, labeled thyroid glands were cultured in vitro in medium containing 1 mM mononitrotyrosine. The rate of the hydrolysis of labeled thyroglobulin was measured as the proportion of 131I-labeled iodotyrosines and 131I-labeled iodothyronines recovered at the end of culture and was used as an index of thyroid secretion. TSH in vivo stimulated the rate of thyroglobulin hydrolysis for 6 h, with a peak occurring after 2 h. The diabetic mice had a diminished response to TSH, which improved on treatment with insulin. The addition of TSH and insulin to the culture medium significantly increased the rate of thyroglobulin hydrolysis in glands of diabetic mice over that resulting from the addition of dibutyryl cAMP alone. The generation of thyroidal cAMP in response to TSH was higher in diabetic mice than in controls. The rise in plasma T4 and T3 2 h after the administration of TSH was less in diabetic mice than in control mice or diabetic mice treated with insulin. Our studies, therefore, indicate that the thyroidal response to TSH is decreased in diabetes mellitus. The defect appears to be at a step beyond the generation of cAMP.     

Morphological and biochemical responses of cultured thyroid cells to thyrotropin

In the thyroid gland, TSH stimulates cAMP formation, exocytosis of precursor (noniodinated) thyroglobulin, endocytosis of thyroglobulin, and proteolytic processing of the thyroglobulin to form thyroid hormones. In this report we describe TSH effects on cAMP levels, microtubules, microfilaments, myosin fibrils, and the morphology of cultured thyroid follicle cells. The cells were normally cultured in the presence of 10 mU/ml TSH, and fresh TSH produced no stimulation when assayed for cAMP production in a 15-min assay. When such cells were cultured for up to 72 h in the absence of TSH and then assayed for cAMP production, the basal levels were much reduced, but fresh TSH stimulated cAMP levels half-maximally at 1 mU/ml and up to 50-fold at 20 mU/ml. Microtubules, myosin fibers, and microfilaments were demonstrated by indirect immunofluorescent staining. Fluorescent staining of fibers was observed in cells fixed before lysis and in cells lysed before fixation. In control cells grown without hormone, microtubules originated near the nucleus and extended to the cell periphery. Myosin-containing fibrils traversed the cell or radiated from foci. Microfilaments spanned the cell in a stress fiber pattern. After incubation with 20 mU/ml TSH and 4 mM isobutylmethylxanthine (IBMX) for 10-20 min, the microtubules in up to half of the cells appeared altered and more granular, and the cell periphery was scalloped. After 15-30 min with TSH and IBMX, normal myosin fibers were replaced with a fine lattice-work, peripheral staining disappeared, and the proportion of nonfibrous myosin increased. Stress fibers demonstrated with antibody to actin also disappeared, and the peripheral structures observed in normal cells became fragmented. Incubation with forskolin or TSH and IBMX for 2-3 h resulted in arborization of 30-60% of the cells that contained bundles of microtubules, myosin fibers, or microfilaments into dendrite-like processes and increased staining near the nucleus. At 5 h, more than 80% of the cells were arborized. These morphological changes were less pronounced with IBMX alone and minimal with TSH alone. The time course of cAMP levels observed basally or after TSH, forskolin, or TSH and IBMX was consistent with the relative effects of these agents on arborization. These studies are consistent with effects of cAMP on microtubules, myosin-containing fibrils, and microfilaments and may provide a basis for the morphological response to TSH.     

TSH secretion in Cushing's syndrome: relation to glucocorticoid excess, diabetes, goitre, and the 'sick euthyroid syndrome'

Thyrotrophin (TSH) secretion was studied in 63 patients with Cushing's syndrome (53 patients with pituitary dependent Cushing's disease, eight with adrenocortical tumours, and two with the ectopic ACTH syndrome). Prior to treatment, TSH response to 200 micrograms of TRH intravenously was significantly decreased compared to controls; TSH response was 'flat' (increment less than 2 mU/l) in 34 patients (54%). Patients with a flat response to TRH had significantly higher morning and midnight cortisol levels than patients with a TSH response of 2 mU/l and more; this was not due to differences in serum thyroid hormone levels. Basal TSH, TSH increment after TRH, and stimulated TSH value, but not serum triiodothyronine, were correlated with cortisol measurements (0800 h serum cortisol, midnight cortisol, and urinary free corticoid excretion). After exclusion of 40 patients with additional disease (severe systemic disease, diabetes mellitus, or goitre), cortisol-TSH correlations were even more pronounced (r = -0.73 for midnight cortisol and stimulated TSH levels), while in the patients with additional complications, these correlations were slight or absent. Successful treatment in 20 patients was associated with a rise in thyroid hormone levels and the TSH response to TRH. These results indicate that (1) the corticoid excess but not serum T3 is the principal factor regulating TSH secretion in Cushing's syndrome, (2) a totally flat response to TRH is rare, and (3) TSH suppression and lower than normal serum thyroid hormone levels are reversible after treatment. Since factors like severe systemic disease, diabetes mellitus and goitre also affect TSH secretion, they tend to obscure the statistically significant correlations between cortisol excess and TSH secretion.     

Regulation of thyroid ornithine ornithine decarboxylase (ODC) by thyrotropin. I. The rat.

We studied the effects of TSH on rat thyroid ornithine decarboxylase (ODC) activity. After 1 day of goitrogen treatment, there was an abrupt fall in serum triiodothyronine (T3) a rise in circulating TSH, and a dramatic increase in thyroid ODC activity. Despite the continued rise in TSH and progressive increase in thyroid gland size with further treatment, thyroid ODC activity declined on the third day and remained at submaximal levels. Thyroid ODC activity was also stimulated in a dose-related manner by administration of exogenous TSH. Little TSH effect was noted before 3 h. Maximal ODC activity occurred between 4 and 5 h. The TSH stimulation of ODC could be inhibited by pretreatment with actinomycin D or cycloheximide, suggesting that the increase in ODC activity requires new RNA and protein synthesis. Although pretreatment with agents that alter microtubule structure (e.g., colchicine and vinblastine) prevent stimulation of ODC activity by TSH, additional data suggest, but do not confirm, that hrmone secretion and ODC activation may be dissociable. Further studies were undertaken to determine whether cyclic AMP (cAMP) or prostaglandins played any role in the regulation of thyroidal ODC activity. Dibutyryl cAMP, alone, or together with aminophylline, did not stimulate thyroidal ODC activity in dosages which concomitantly stimulated adrenal enzyme activity. Likewise, prostaglandin E2 (PGE2) did not stimulate thyroidal ODC activity, but did stimulate adrenal enzyme activity in a dose-related manner. However, pre-treatment of rats with inhibitors of prostaglandin synthesis prevented the activation of thyroidal ODC BY TSH. One inhibitor, indomethacin, attenuated the TSH stimulation of enzyme activity in a dose-related manner. Indomethacin pretreatment also resulted in approximately a 10-fold decrease in thyroidal prostaglandin levels. Exogenous PGE9, in dosages as high as 500 pg, did not overcome the inhibitory effect of indomethacin on ODC activation. Although the precise role for endogenous prostaglandins remains to be defined, it does appear that a reduction in thyroidal prostaglandins prevents activation of the enzyme by TSH.     

Inhibition of TSH activation of human cultured thyroid cells by tumor necrosis factor: an explanation for decreased thyroid function in systemic illness?

Thyroidal economy in systemic nonthyroidal illness (the sick euthyroid syndrome) is marked by reductions in both thyroid function and peripheral T4 to T3 conversion, presumed to reflect a homeostatic mechanism to conserve energy. TSH levels tend to be normal in these patients, and the mechanism underlying reduced thyroidal secretion is unknown. Since increased blood levels of tumor necrosis factor (TNF) are found in many of the conditions associated with the sick euthyroid syndrome, we hypothesized that TNF might affect the function of the thyroid gland. We, therefore, explored the effects of TNF on TSH stimulation of the thyroid, employing a human thyrocyte cell culture model. Cells were incubated with various concentrations (0-1000 pg/mL) of recombinant human TNF-alpha and bTSH (1 mU/mL), with measurement of secreted thyroglobulin (Tg) and cyclic AMP (cAMP) as the end points of stimulation. TNF had no effect on either basal or TSH-stimulated cAMP generation, but significantly blunted TSH-stimulated Tg secretion. No loss of cell viability and growth was observed based on trypan blue exclusion and thymidine incorporation by cells. These studies demonstrated an inhibitory effect on TNF on human thyrocytes in concentrations comparable to blood levels seen in humans during systemic illness. We conclude that increases in serum TNF may be responsible for reduced thyroid function in patients with the sick euthyroid syndrome.     

Thyrotropin action is impaired in the thyroid gland of old rats.

Aging in rats is characterized by low plasma concentrations of thyroid hormones with unchanged levels of TSH, suggesting an altered TSH action in addition to the impaired regulation of TSH secretion. To evaluate TSH action we determined TSH binding to thyroid membranes of young and old male rats (3-4 and 24-26 months of age), as well as the activity of adenylate cyclase in basal and stimulated conditions. Saturation analyses of [125I]-bTSH to thyroid membranes in the presence of increasing quantities of unlabelled bTSH (0.03-100 mU) show two types of binding sites, one of high affinity (Ka 1.5 10(9) mol l-1) the other of lower affinity (Ka 1.2 10(8) mol l-1), which are similar in both age groups. The number of TSH binding sites of high affinity is less in old rats than in young rats (7.6 +/- 0.9 vs 14.8 +/- 1.1 TSH mU/mg protein, N = 11 and 10 respectively, p less than 0.001), whereas the number of binding sites of low affinity is not significantly different (76.0 +/- 8.2 vs 99.1 +/- 9.0 TSH mU/mg protein). The activity of adenylate cyclase determined in basal conditions is similar in both old and young rats (1.11 +/- 0.12 vs 1.04 +/- 0.9 nmol cAMP/2 h x mg/protein). TSH (10 mU) induced a significant increase in cAMP formation with the thyroid membranes from young rats but not with those from old rats. In contrast, the stimulation of cAMP formation by GTP (2 mmol/l) or forskolin (10 mmol/l), two direct stimulators of adenylate cyclase, is similar in both groups of rats (200% and 250%, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of intrathyroidal metabolism of thyroxine on thyroid hormone secretion: increased degradation of thyroxine in mouse thyroids stimulated chronically with thyrotrophin

Mice were infused continuously with graded doses of bovine TSH (bTSH) and changes in plasma concentrations of bTSH and T4 were measured. Then mice infused with 100 mU TSH per day were sacrificed on days 0, 1, 3 and 5 and their thyroids were excised to determine in vitro secretion of T4, T3 and rT3 during 3 h of incubation. At the end of the incubation, thyroidal contents of T4, T3 and rT3 were also determined after pronase digestion. Plasma bTSH levels were increased on day 1 to a level of 110 microU/ml and remained unchanged thereafter. Plasma T4 concentrations increased approximately 2-fold on day 1, but decreased to initial levels on days 3 and 5. Changes in T4 secretion in vitro paralleled those in plasma T4 concentrations; T4 secretion increased 2-fold on day 1, and decreased to the pre-TSH levels on days 3 and 5. In contrast, T3 secretion increased throughout the experimental period. The T3/T4 ratio in thyroidal secretion in vitro was the same as that in thyroidal contents on days 0 and 1 of TSH infusion, but the former was significantly greater than the latter on days 3 and 5. PTU (5.9 X 10(-5) M), a known inhibitor of T4 deiodination, added to the incubation media did not affect T4 secretion on days 0 and 1, but increased T4 secretion on days 3 and 5 to the level of day 1, but did not affect T3 secretion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of in vivo triiodothyronine and long acting thyroid stimulator (LATS) administration on the vitro thyroid cAMP response to thyrotrophin and LATS

The present study was undertaken to examine the effects of prolonged in vivo treatment with T3 and long acting thyroid stimulator (LATS) on in vitro responsiveness of mouse thyroid cyclic AMP to thyrotrophin (TSH) and LATS-immunoglobulin G (IgG). In control mice, thyroid cAMP concentrations after incubation with normal-IgG (10 mg/ml) for 2 h, TSH (10 mU/ml) for 10 min and LATS-IgG (10 mg/ml) for 2 h were 1.25 +/- 0.11 (mean +/- SE) (n = 5), 15.87 +/- 3.47 (n = 6) and 2.17 +/- 0.25 pmoles/mg wet weight (n = 6), respectively. In mice given T3 (5 micrograms/ml) in drinking water for 5 days, thyroid cAMP concentrations after an incubation with TSH were reduced by 50%, as compared to those of the control mice. They were also decreased in mice injected ip with 5 mg of LATS-IgG (1000%/5 mg in the McKenzie bioassay) daily for 5 days. Combined treatment with T3 and LATS decreased the cAMP response to TSH only to the same extent as did T3 alone, indicating that the inhibitory effects of T3 and LATS were not additive. Similar findings were observed with the thyroid cAMP response to LATS-IgG in vitro; either T3 or LATS treatment in vivo decreased cAMP response to LATS-IgG in vitro, but combined treatment with T3 and LATS did not cause further inhibition as compared with T3 or LATS treatment alone.(ABSTRACT TRUNCATED AT 250 WORDS)