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Binding of 125I-labelled bovine TSH to crude membrane fractions of human thyroid tissue was a saturable, hormonally specific process which yielded non-linear Scatchard plots with limiting affinities of approximately 109 and 107l/mol. Binding activity in membranes was soluble in Triton X-100, was inhibited specifically by immunoglobulins from patients with Graves's disease, and was increased by the beta-blocking drug, propranolol. In contrast, purified nuclear preparations showed a predominance of lower affinity binding, and their binding activity was insoluble in Triton and insensitive to immunoglobulins from patients with Graves's disease and to propranolol. Tryptic digestion liberated only low affinity binding activity from membranes or nuclei. It was concluded that human thyroid tissue contains independent classes of TSH-binding sites, which differ in their chemical, immunological and hormone-binding properties.
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SUMMARY
The changes in the kinetics of 131I131I− accumulation accompanying the thyrotrophin (TSH)-induced decrease in the tissue/medium concentration ratio (T*/M*) in propylthiouracil-blocked guinea-pig thyroid slices in vitro have been investigated. 131I− was eluted in serial 2 ml fractions from prelabelled 10 mg thyroid slices incubated for 10 h with TSH. Retrapping of 131I− from the extrafollicular space was abolished by including ClO4 − in the elution medium. In the presence of ClO4 −, departures from 1st order kinetics were detected by the analysis of regression of successive estimates of instantaneous exit rate, though not by the analysis of regression of log e T t */T 0 *. It was predicted that initial exit data would be more representative of exit rates under equilibrium conditions. TSH gave increases in the fractional exit rate (K tm ) of similar order to those required to account for the decrease in T*/M*.
The data support the hypothesis that an increase in 131I− exit rate mediates the decrease in T*/M* induced by TSH.
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SUMMARY
Particulate fractions of guinea-pig thyroid homogenate contained a single class of receptors which bound 125I-labelled bovine thyrotrophin (TSH) by a saturable, reversible process with an affinity constant of 2 × 109 1/mol. The binding process was specific for TSH, and corresponded with the activation of adenylate cyclase. Cleavage of hormone—receptor bonds by treatment with lyotropic agents resulted in the release of unchanged labelled TSH. The radioligand receptor assay system was sensitive to 0·015 mu. TSH. Bovine or mouse thyroid showed reduced binding affinity with correspondingly reduced sensitivity.
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SUMMARY
Immunoglobulin G (IgG) prepared from sera containing the long-acting thyroid stimulator (LATS) inhibited the receptor binding of 125I-labelled thyrotrophin (TSH) in a particulate fraction of guinea-pig thyroid homogenate. This inhibition was shown to involve a binding interaction between IgG containing LATS and the receptor with a diminution in the number, but not affinity, of sites available for binding of TSH. Studies of dissociation kinetics and gel filtration of receptor—TSH complexes indicated that IgG containing LATS did not combine with receptors occupied by TSH. The data provide evidence that LATS and TSH bind to the same receptor site.
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SUMMARY
125I-Labelled thyrotrophin (TSH) showed saturable binding to hyperplastic guinea-pig thyroid slices in vitro, but not to kidney, adrenal, liver, testis or salivary gland slices. Two populations of binding sites were demonstrated: high order sites showed an affinity constant of 3·8 × 1081/mol and a capacity of 8 × 103 molecules of TSH/cell; low order sites, affinity constant of 2·9 × 1071/mol and capacity of 8 × 104 molecules/cell. The highorder sites saturate at about 1 mu./ml, approximately the level producing maximal in-vivo and in-vitro responses. Binding of 125I-labelled TSH was reversible, the released hormone appearing substantially unchanged as assessed by chromatographic behaviour and antiserum binding. Release of unlabelled TSH from pre-incubated thyroid slices was demonstrated by bioassay. Bovine thyroid slices were found to have a lower capacity for binding than hyperplastic guinea-pig slices.
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SUMMARY
The presence of thiocyanate ion in human and rat serum has been shown to account entirely for the non-specific activity of sera in an in-vitro bioassay for thyrotrophin. Thiocyanate was identified by its chromatographic behaviour on Sephadex G-10, G-15 and G-25, and by the ferric nitrate and cobalt nitrate tests. Cigarette smoking increased mean serum thiocyanate levels (as NaSCN) from 0·2 to 0·56 mg/100 ml. It is suggested that serum thiocyanate levels are sufficient to inhibit significantly iodide trapping in vivo and that these findings may be relevant to the non-specific responses observed with other in-vitro bioassays based on radioiodine dynamics.
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SUMMARY
The effect of methallibure (ICI 33,828), a non-steroidal pituitary inhibitor, on serum and pituitary thyroid-stimulating hormone (TSH) levels has been investigated.
A biphasic action of the drug on serum TSH levels was observed, the greatest falls occurring with the lowest doses (2mg/day). Increasing dose and period of administration induced progressive decreases in pituitary TSH content. These results are interpreted in terms of three actions on the thyroid—pituitary system: (1) inhibition of the release of TSH from the pituitary, (2) inhibition of TSH synthesis evident only at higher doses, and (3) a thyroid-blocking action, which is also only observed at the higher dose levels, with consequent pituitary stimulation via the thyroid—pituitary feedback mechanism. Effects upon body weight and weight of endocrine organs are reported, that upon the seminal vesicles being the most marked.
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ABSTRACT
Veratridine, a sodium channel agonist, depolarized cultured thyroid cells and increased the secretion of radioiodine from the organically bound pool. These effects were similar to those of TSH. Depolarization of the cells by increasing the potassium concentration of the medium failed to promote secretion, indicating that the sodium influx, rather than the depolarization itself, mediated the response. Veratridine, like TSH, also acutely reduced the cells' iodide uptake and inhibited the iodide transport pump. Unlike TSH, however, veratridine reduced, rather than increased, the fractional exit rate of iodide anion from the unbound pool. The data are consistent with the hypothesis that a sodium influx mediates some but not all of the actions of TSH on the thyroid gland, including the stimulation of secretion of thyroid hormones.
J. Endocr. (1986) 110, 459–466
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ABSTRACT
Thyrotrophin stimulated release of radioiodine from the organic iodine pool of cultured porcine thyroid cells. The response was well developed within 2–4 h of incubation. Inhibition of the Na+/K+ pump with ouabain or incubation in sodium-free medium inhibited the response to TSH. The magnesium content of cultures was reduced by ouabain, and increasing the magnesium concentration of the medium to 10 mmol/l reversed the inhibition of the TSH response by ouabain. After prolonged incubation (4–6 h), ouabain in magnesium-enriched medium stimulated release of radioiodine. Its effects were not additive with those of TSH. Incubation for 4–6 h in media of reduced sodium concentration (34 mmol/l) also stimulated release. Sodium-free medium alone did not alter basal release rates, but magnesium enrichment of sodium-free medium promoted release after 4 h of incubation. It was concluded that the previously reported inhibition of the TSH response in thyroid tissue by ouabain or sodium-free medium was due to secondary derangements of cellular function rather than to a specific blockade of the secretomotor signal. The data are consistent with the hypothesis that a reduction in the sodium ion electrochemical gradient across the cell membrane mediates the secretomotor effect of TSH.
J. Endocr. (1987) 112, 399–405
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ABSTRACT
Cultured porcine thyroid cells did not reassociate into functional follicles in the presence of TSH unless the initial seeding density was adequate. At 0·2 × 106 cells/35 mm diameter culture dish the cells rapidly formed a monolayer even in the presence of TSH (128 μu./ml), and radioiodide uptake was not significantly increased compared with that in control cells. Seeding densities of 1–3 × 106 cells/dish resulted in cultures which responded to TSH with follicular development and increased radioiodide uptake. A cell-free membrane fraction of thyroid homogenate restored the ability of cultures seeded at low densities to respond to TSH with development of follicular morphology and increased radioiodide uptake. Delaying the addition of TSH by 48 h markedly reduced the stimulation of follicular development and radioiodide uptake of cultures. Addition of membrane fractions, or an alkali-soluble fraction of membranes, at zero time improved the responses to TSH added after a 48-h delay. It was concluded that maintenance of differentiation and of TSH-responsiveness in cultured thyroid cells was influenced by cell–cell contact.
J. Endocr. (1987) 113, 223–229
