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SUMMARY
Using McKenzie's [1958b] modification of Adams & Purves's [1955] method for bioassay of thyrotrophin, which depends on the release into the circulating blood of radio-iodine from thyroid glands of mice, an attempt was made to measure thyrotrophin in human serum. Two types of response were encountered. The first, characteristic of hypothyroidism, resulted in a peak increase in blood radioactivity 2 hr after injection and resembles the response to standard thyrotrophin preparations. The second response, characteristic of hyperthyroidism, was delayed and the blood radioactivity did not reach a peak until 12 hr after the serum injection. The incidence of the two types of response has been investigated and several additional differences between the delayed response and the response to thyrotrophin extracted from pituitary tissue have been established.
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SUMMARY
The binding activity of fresh human thyroid homogenates for the long-acting thyroid stimulator (LATS) was shown to be principally associated with the particulate fraction. LATS binding activity (LAA) could be partially released into the soluble fraction by freezing and thawing of thyroid homogenates. By delaying freezing and thawing until the particulate fraction had been washed repeatedly it was possible to obtain highly purified preparations of LAA.
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SUMMARY
The response to the long-acting thyroid stimulator (LATS) but not to bovine thyroid-stimulating hormone (TSH) was reduced by soluble fractions from human, ovine, bovine and porcine thyroids. A significant increase in the capacities of human, ovine and bovine thyroidal soluble fractions to reduce the response to LATS occurred upon freezing and thawing of thyroidal homogenates. In human thyroids this change took place predominantly in the 4 S peak, which was the most active of the soluble fractions from frozen human thyroid tissue. However, in ovine and bovine thyroids, the change could be accounted for mainly by components of the 19 S peak, this being also the most active of the soluble fractions of frozen animal thyroids in reducing the response to LATS.
Whereas human LATS-binding activity (LAA) was destroyed by heating at 56 °C for 1 h the capacities of ovine, bovine and porcine thyroidal soluble fractions (or their 19 S components) to reduce the response to LATS were not affected by this treatment.
The interaction between human LAA and LATS can be reversed by 2 m-NaSCN, which also destroys LAA. However, NaSCN had no detectable effect on the capacities of soluble fractions (or their 19 S components) from ovine, bovine and porcine thyroids to reduce the response to LATS. The reduction of the response to LATS by very high concentrations of 4 S components, from frozen ovine thyroidal homogenate, was partially reversed by 2 m-NaSCN. Thus ovine thyroids may be a source of a substance with the properties of human LAA, but with a yield of approximately 10% of that obtained from human thyroid tissue.
It was concluded that ovine, bovine and porcine thyroidal soluble fractions reduced the response to LATS mainly by mechanisms other than binding to LAA.
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SUMMARY
In the in-vitro assay of Brown & Munro (1967) thyroid-stimulating hormone (TSH) increased the release of radioactive iodine from mouse thyroid glands labelled with 131i during life. Paper chromatography showed that TSH increased the 131I-labelling of thyroxine and tri-iodothyronine both in the culture medium and in hydrolysates of the thyroids.
Cyclic 3′,5′-adenosine monophosphate (cyclic AMP) also increased 131I release in this assay and increased the 131I-labelling of thyronines in the culture medium. The effects on thyroid hydrolysates were less striking.
Theophylline potentiated the influence of TSH and cyclic AMP in the assay and, by itself, increased 131I release and the labelling of iodothyronines in the thyroid without altering the distribution of 131I in the culture medium.
The implications of these results are discussed.
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SUMMARY
A new in vitro assay for thyroid-stimulating hormone (TSH) is described. The parameter of TSH action is the discharge of radioactive iodine from mouse thyroid glands labelled with 131I in vivo. The assay is sensitive to human TSH and gave consistent results during 1 yr. without seasonal variation. A potent preparation of long-acting thyroid stimulator gave a dose-response line parallel with human TSH. Fresh human serum was toxic to the assay preparation so that circulating TSH levels cannot be measured.
Search for other papers by K. J. DORRINGTON in
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SUMMARY
1. A dry powdered standard for the long-acting thyroid stimulator (LATS) has been prepared and shown to be satifactory when tested, at weekly intervals, over a period of 18 months.
2. The difficulties encountered in earlier attempts to establish the standard are discussed.
3. Although supplies of the LATS standard are limited it is hoped that there will be sufficient to supply it to other laboratories so that substandards may be set up by the methods described in this paper.
Search for other papers by PAT KENDALL-TAYLOR in
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SUMMARY
The effects of dibutyryl cyclic 3′,5′-adenosine monophosphate (DBc-AMP) on the mouse thyroid gland have been investigated in the in-vitro assay of Brown & Munro (1967). The distribution of 131I-labelled compounds in the glands and the supporting medium have been analysed by thin-layer chromatography and the changes induced by cyclic 3′,5′-adenosine monophosphate (c-AMP), DBc-AMP or thyroid-stimulating hormone (TSH) compared.
The release of 131I was increased when the glands were incubated with DBc-AMP, c-AMP or TSH. The potency of DBc-AMP was approximately 50 times that of c-AMP on a basis of molarity. Like TSH, DBc-AMP increased the proportion of iodothyronines in the system as a whole, whereas c-AMP had little effect. The possible explanations for this are discussed.
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SUMMARY
The prolonged action of long-acting thyroid stimulator (LATS) in the McKenzie assay could be related to the slow clearance of LATS from the blood of assay mice.
Injection of soluble thyroid extracts before or after LATS reduced the assay response. This corresponded with the inactivation of circulating LATS activity by the formation of a complex which was then rapidly removed from the blood.
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ABSTRACT
Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1·4 × 109 l/mol); the density of binding sites was low compared with the TSH receptor. At 37 °C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0·5 nmol/l) using 0·5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity.
J. Endocr. (1984) 102, 57–61
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ABSTRACT
Human thyroid epithelial cells in monolayer culture were found to release radioimmunoassayable insulinlike growth factor-I (IGF-I) over a 48-h culture period in serum-free medium. In the presence of supraphysiological concentrations of TSH (1–100 mU/ml) known to be inhibitory to DNA synthesis by human thyroid cells, the release of IGF-I was found to be inhibited in six thyroid cultures studied. In only one out of the six was IGF-I release increased in the presence of physiological mitogenic concentrations of TSH (0·1–100 μU/ml). Human thyroid fibroblasts, established by long-term culture of thyroid epithelial cells under fibroblast-selective conditions, also secreted IGF-I which was unaffected by the presence of TSH at both low and high concentrations.
Using a monoclonal antibody against human IGF-I, monolayer cultures of both human thyroid epithelial cells and human thyroid fibroblasts showed positive immunocytochemical staining for IGF peptide. However, fixed sections of intact thyroid tissue only showed positive staining for IGF peptide associated with the fibrous layers surrounding the thyroid follicle, with no staining of the follicular epithelial cells.
The growth of human thyroid epithelial cells was also found to be increased by IGF-I (25–100 ng/ml) added in medium plus 1 % fetal calf serum as assessed by the incorporation of [3H]thymidine into DNA. In the presence of a monoclonal antibody to IGF-I the increase in [3H]thymidine uptake in response to IGF-I was abolished as was that seen in response to TSH.
This study indicates a possible paracrine/autocrine role of IGF-I in the regulation of human thyroid epithelial cell proliferation by interaction with TSH.
Journal of Endocrinology (1989) 123, 495–500
