TSBAb (TSH-stimulation blocking antibody) and TSAb (thyroid stimulating antibody) in TSBAb-positive patients with hypothyroidism and Graves' patients with hyperthyroidism.

There are two types of TSH receptor antibodies (TRAb); thyroid stimulating antibody (TSAb) and TSH-stimulation blocking antibody (TSBAb). TSAb causes Graves' hyperthyroidism. TSBAb causes hypothyroidism. Both TSAb and TSBAb block TSH-binding to thyroid cells as TSH receptor antibodies (TRAb). TSBAb-positive patients with hypothyroidism and Graves' patients with hyperthyroidism may have both TSBAb and TSAb. We studied TSBAb and TSAb in 43 TSBAb-positive patients with hypothyroidism and in 55 untreated Graves' patients with hyperthyroidism. TSBAb-activities were expressed as percentage inhibition of bovine (b) TSH-stimulated cAMP production by test IgG. Two formulas were used to calculate TSBAb-activities; TSBAb-A (%) = [1 - (c - b)/(a - b)] x 100 and TSBAb-B (%) = [1 - (c - d)/(a - b)] x 100, where a: cAMP generated in the presence of normal IgG and bTSH, b: cAMP generated in the presence of normal IgG, c: cAMP generated in the presence of test IgG and bTSH, and d: cAMP generated in the presence of test IgG. TSAb (%) = [d/b] x 100. All of the 43 TSBAb-positive patients with hypothyroidism had strongly positive TSBAb-A and -B. Some of them had weakly positive TSAb (<240%). All 55 untreated Graves' patients had positive TSAb (205-2509%). Some of them had both TSAb and TSBAb. TSBAb-positive patients with hypothyroidism had a limited distribution of TSBAb- and TSAb-activities (TSBAb-A + 75 - + 103%, TSBAb-B + 87 - + 106%, TSAb 92-240%), but Graves' patients with hyperthyroidsim had a wide distribution of TSAb- and TSBAb-activities (TSAb 205-2509%, TSBAb-A - 158 - + 43%, TSBAb-B - 14 - + 164%). TSBAb-A ignores TSAb activity in serum, and might give low TSBAb activity. However, TSBAb-A clearly differentiates TSBAb-positive patients with hypothyroidism from Graves' patients with hyperthyroidism; thus, we favor TSBAb-A over TSBAb-B. Some of TSBAb-positive patients with hypothyroidism and Graves' patients with hyperthyroidism have both TSBAb and TSAb.     

Non-suppressed thyroidal radioactive iodine uptake (RAIU) in thyrotoxic phase in a case of subacute thyroiditis with thyroid-stimulating antibodies (TSAb).

In this paper, we report a 49-year-old female with subacute thyroiditis who had thyroid-stimulating antibodies (TSAb) and thyroid-stimulation-blocking antibodies (TSBAb) in serum. Although she was in the thyrotoxic phase and TSH was suppressed in May, 1990, her radioactive iodine uptake (RAIU) was not suppressed (35.5%) and a thyroid scan disclosed a diffuse goiter with no defect. Serum assays revealed the presence of TSAb, but TSBAb were negative. In August, 1990, the right lobe became undetectable by thyroid scan when the RAIU was 20.7% with the TSH level remaining suppressed. At that time, TSAb were negative, while TSBAb were positive. When the RAIU was 31.1% in October, 1990, both thyroid lobes became visible and the TSH level was normalized. TSBAb became negative, and although TSAb reappeared it later became undetectable. These results indicate that the changes in the patient's thyroid scan and RAIU were attributable to the presence of TSAb.     

Similarity and dissimilarity between clinical and laboratory findings, especially anti-thyrotropin receptor antibody in ophthalmic Graves' disease without persistent hyperthyroidism and hyperthyroid Graves' disease

The aim of this study was to investigate thyroid states, significance of anti-TSH receptor antibodies and the clinical courses of patients with euthyroid Graves' ophthalmopathy. The clinical and laboratory finding of 30 patients with euthyroid Graves' ophthalmopathy were briefly as follows: 1) normal sized thyroid or small goiter; 2) negative or weakly positive thyrotropin binding inhibitor immunoglobulin (TBII); 3) normal thyroid [99 m-Tc] pertechnetate uptake; and 4) frequent observations of low serum TSH values. Besides TBII, thyroid stimulating antibody (TSAb) was measured under low salt and isotonic conditions using FRTL-5 rat thyroid cells. Both TBII and TSAb titers were lower in euthyroid Graves' ophthalmopathy than in hyperthyroid Graves' disease. Serum TSH levels frequently became low in patients considered as euthyroid upon the first examination as well as in Graves' patients in remission, reflecting preceding or mild hyperthyroidism. In follow-up studies, these patients with mildly elevated thyroid hormone levels and low TSH levels seldom reached a state of persistent hyperthyroidism, when TBII was negative or only weakly positive.     

Detection of thyroid-stimulating antibody in patients with inflammatory thyrotoxicosis.

The detection of thyrotropin-binding inhibitory immunoglobulins (TBII) and/or thyroid-stimulating antibody (TSAb) has been reported in some patients with painless thyroiditis (PT) or subacute thyroiditis (SAT). However, its mechanism is unknown. TBII and TSAb measured using cultured FRTL-5 thyroid cells were evaluated in 18 patients with PT, 11 patients with SAT and a patient with SAT-like symptoms. In PT, we detected both TBII and TSAb activities in only 1 patient. This case had first come to our attention with subclinical hypothyroidism and had already had weakly positive TSAb activity (205.9%) 1 year before the present onset of PT. This patient had a transient thyrotoxicosis with a low uptake (24 h) of 123I (4.3%) and 821.0% TSAb activity, and subsequently developed a transient subclinical hypothyroidism. Even after 2 years, she still had positive TSAb activity (382.3%). In SAT, TBII and TSAb activities were not detected during the courses of any patients. A patient with transient thyrotoxicosis, who had a high uptake (30 min) of 99mTc (5.6%) and SAT-like symptoms (painful tenderness on right thyroid lobe and markedly accelerated erythrocyte sedimentation rate), showed positive activities of TBII (34.9%) and TSAb activity (1,366.9%). Histological findings by thyroid needle biopsy performed in the thyrotoxic phase showed coexistence of granulomatous inflammatory changes and hyperplasia with papillary folds of some residual follicular cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitive assay to detect thyroid stimulating antibody (TSAb) in the presence of thyroid stimulation blocking antibody (TSBAb) in serum.

The detection of thyroid stimulating antibody (TSAb) activity in the presence of thyroid stimulation blocking antibody (TSBAb) in Graves' serum is difficult because TSBAb blocks TSAb activity. We recently demonstrated that polyethylene glycol (PEG) augments TSAb activity in porcine thyroid cells (PTC) assay. This PEG-induced augmentation makes it possible to develop a sensitive assay to detect TSAb in the presence of TSBAb. We studied the effects of PEG on TSAb- and TSBAb-activities in PTC using 4 different preparations of the samples; (1) crude IgG using PEG 22.5% precipitated fraction (PF) from Graves' serum (0.2 ml), (2) crude IgG using PEG 12.5% PF, (3) serum (50 microl), and (4) serum (50 microl) in the presence of 5% PEG (final). When the effects of PEG on TSAb activity using crude IgG were examined, PEG 22.5% PF showed significantly higher TSAb activity than PEG 12.5% PF as reported previously. The augmentative effect of PEG on TSAb activity was also observed by the addition of 5% PEG to serum. We also demonstrated that PEG augmented TSAb-activities even in TSBAb-positive serum by two methods (crude IgG using PEG 22.5% PF and the addition of 5% PEG to serum). TSBAb activities were expressed by two calculation methods (A= [1 - (a - b)/(c - d) x 100] and B = [1 - (a - d)/(c - d) x 100], where a is cAMP produced in the presence of bTSH and patient's IgG, b is cAMP produced in the presence of patient's IgG, c is cAMP produced in the presence of bTSH and normal IgG, and d is cAMP produced in the presence of normal IgG). In the presence of TSAb, the values of A method were always higher than those of B method, since TSAb stimulated cAMP synthesis. We have developed two sensitive methods to detect TSAb even in the presence of TSBAb in serum using PEG; 1) incubation of crude IgG using PEG 22.5% PF from serum (0.2 ml), and 2) co-incubation of 5 % PEG with test serum (50 microl).     

A case of Graves' disease and papillary thyroid carcinoma with predominant production of thyroid-stimulation-blocking antibodies (TSBAb) persisted after total thyroidectomy.

A 42-year-old female with Graves' disease and papillary thyroid carcinoma with lung metastasis was referred to our hospital. After treatment of thyrotoxicosis with methimazole and Lugol's solution, she underwent total thyroidectomy. She was then given 131I twice to treat lung metastasis. However, 131I uptake into the lung was not clear in the scintigram. Both thyroid-stimulating antibodies (TSAb) and thyroid-stimulation-blocking antibodies (TSBAb) were detected in her sera before and after the treatments. Compared with TSAb activities, TSBAb activities were extremely high. Changes in the titers of these two antibodies were not clear after total thyroidectomy. These results indicate that lymphocytes outside the thyroid gland are the major source of TSAb and TSBAb in this patient.     

Lack of interaction in recombinant human FSH receptor and both TSAb and TSBAb

Since cross-reactivity of TSH with the human FSH receptor has been reported, in this study we tested the effect of thyroid-stimulating antibody (TSAb) and thyroid stimulation-blocking antibody (TSBAb) on Chinese hamster ovary cells expressing human FSH receptor (CHO-hFSH-R cells). We examined the TSBAb activity of sera from hypothyroid patients who had a positive TBII to determine whether these sera also block the effect of FSH on CHO-hFSH-R cells. Although human FSH I-3 (0.25-16 ng/ml) stimulated the production of intracellular cAMP in CHO-hFSH-R cells with dose-responsive manner, neither TSAb nor TSBAb had such an effect on the cells.     

Role of N-terminal region of the thyrotropin (TSH) receptor in signal transduction for TSH or thyroid stimulating antibody.

To identify the site(s) on the thyrotropin (TSH) receptor that interacts with TSH or thyroid stimulating antibody (TSAb), we examined the effect of the synthetic TSH receptor peptide (termed N2 peptide, No. 35-50) on the cAMP accumulation induced by TSH or TSAb. Preincubation of bovine TSH with N2 peptide resulted in a significant and dose-dependent decrease in cAMP accumulation. This decrease was not observed when bovine TSH was preincubated with P1 peptide, which was used as a control (No. 398-417). In contrast, the N2 peptide did not affect TSAb activity in immunoglobulin fractions from three TSAb-positive patients with Graves' disease. P1 peptide also had no effect on TSAb activity. These results suggest that the N-terminal region of the TSH receptor is important for TSH action, and also that TSAb activity cannot be suppressed only by the application of the synthetic peptide corresponding to the N-terminal region.     

Thyrotropin receptor non-mediated thyroid stimulating immunoglobulin in Graves' disease.

There exists a consensus that hyperthyroid Graves' disease is caused by thyrotropin receptor (TSH-R) autoantibodies. To test the possibility that the TSH-R is the sole antigen for thyroid stimulating antibodies (TSAb), we compared bioactivities of Graves' IgGs between non-thyroid mammalian cells transfected with human TSH-R cDNA and the reference thyroid bioassay. A Graves' IgG with TSH-binding inhibitor immunoglobulin (TBII) activity (89%) markedly stimulated cAMP formation in both CHO-K1 cells transfected with TSH-R cDNA (340 microU/ml of TSH equivalent) and rat thyroid cells, FRTL-5, (410 microU/ml of TSH equivalent). In contrast, a TBII negative (-1.5%) IgG from another patient with Graves' disease showed a strong thyroid stimulating activity (87 microU/ml of TSH equivalent) when FRTL-5 cells were used for the assay. But no stimulating activity was observed in this IgG when CHO-K1 cells transfected with TSH-R cDNA were used, suggesting a possible existence of TSH-R non-mediated thyroid stimulating immunoglobulin in some cases of Graves' disease.     

A synthetic oligopeptide derived from human thyrotropin receptor sequence binds to Graves' immunoglobulin and inhibits thyroid stimulating antibody activity but lacks interactions with TSH.

An 11-residue oligopeptide, P-195, was synthesized to match human thyrotropin (TSH) receptor structure from No. 333 to 343 of amino acid sequence. Preincubation of 5 Graves' IgGs with P-195 up to 10 micrograms resulted in dose-dependent reductions of thyroid stimulating antibody (TSAb) activity. [125I] labeled P-195 was found to bind Graves' IgG. The bound radioactivity correlated significantly with their TSAb activity (N = 25, r = 0.587, p less than 0.01). A peptide having a completely reverse sequence as P-195 did not show such biological activity. The peptide did not affect TSH and thyrotropin binding inhibitor immunoglobulin (TBII) on their receptor binding nor biological activities. P-195 was concluded to have a part of TSAb binding sites.     

Differential effects of growth factors on [3H]thymidine incorporation and [125I]iodine uptake in FRTL-5 rat thyroid cells

We studied the effect of several growth factors on DNA synthesis and function of FRTL-5 rat thyroid cells by simultaneous measurement of [3H]thymidine incorporation and [125I]iodide uptake. Endothelial cell growth factor, fibroblast growth factor, platelet-derived growth factor, and insulin-like growth factor I stimulated thymidine incorporation in a dose-dependent manner without the parallel increase of [125I]iodide uptake. These growth factors had an additive effect with thyroid-stimulating hormone (TSH) on thymidine incorporation, but they inhibited TSH-stimulated iodide uptake. Bombesin stimulated thymidine incorporation and inhibited TSH-stimulated iodide uptake; epidermal growth factor and gastrin-releasing peptide 10 had neither effect. None of the growth factors studied affected iodide uptake in the absence of TSH. Of the growth factors tested, endothelial cell growth factor, fibroblast growth factor, insulin-like growth factor bombesin, and platelet-derived growth factor all share similar differential effects on FRTL-5 cells: stimulation of DNA synthesis, potentiation of the effects of TSH on DNA synthesis, and attenuation of the effects of TSH on cell function. The data suggest that these growth factors may play important roles in regulation of thyroid function.     

Development of chicken antibodies toward the human thyrotropin receptor peptides and their bioactivities.

We have synthesized four peptides (P2, P4, E3 and P1) corresponding to different segments of human thyrotropin (TSH) receptor. We have obtained antibodies by immunizing them to chickens, and antibodies are evaluated for their thyroid stimulating antibody (TSAb), thyroid stimulation blocking antibody (TSBAb) and TSH-binding inhibitor immunoglobulin (TBII) activities. None of the antibodies had TSAb activity. Antibodies against human TSH receptor specific region such as P2 and P4 (P2: No. 372-397, P4: No. 341-358) had TSBAb and TBII activities. Anti-E3 antibody (E3: the third putative extracellular loop, No. 649-661) had only TSBAb activity. Anti-P1 antibody (P1: high homology with pig LH/CG receptor, No. 398-417), however, had none. These results suggest that anti-TSH receptor antibodies to different antigenic epitopes show heterogeneity in their biological activities.     

Clinical usefulness of TSAb assay with high polyethylene glycol concentrations.

We previously demonstrated the stimulatory effect of polyethylene glycol (PEG) on thyroid-stimulating antibody (TSAb)-IgG-stimulated cAMP production (thyroid stimulating (TS) index) in porcine thyroid cell (PTC) assay. In the present study the clinical usefulness of the practical method using high PEG concentrations was examined. TS activity using PEG 22.5% precipitated fraction (PF) was significantly higher compared to standard TSAb activity using 12.5% PF from TSAb-positive serum, but the maximum TS activity was observed with PEG 12.5% PF + 4% PEG or PEG 22.5% PF + 2% PEG. In all cases of untreated Graves' patients, TSAb activity determined by PEG 22.5% PF was higher compared to standard TSAb activity using PEG 12. 5% PF from test serum, but the highest TSAb activity was observed by PEG 12.5% PF + 4% PEG without increased cAMP production to normal serum. TSAb was positive in 85% (40/47), 98% (46/47) and 100% (47/47) of untreated Graves' patients by the method of PEG 12.5% PF, PEG 22.5% PF and PEG 12.5% + 4% PEG, respectively. Increased TSAb activity by PEG 12.5% PF + 4% PEG method was also observed even if the standard TSAb activity using PEG 12.5% PF method was negative in the euthyroid states of Graves' patients during antithyroid drug therapy. The stimulatory effect of PEG on TS activity was not found in other thyroidal diseases [thyroiditis chronica (with high serum TSH), thyroid stimulation-blocking antibody (TSBAb)-positive sera (with low serum TSH), adenomatous goiter, subacute thyroiditis, and thyroid cancer]. The stimulatory effect of 5% PEG on TS activity produced directly by small amounts of Graves' serum (50 microl) was also found, although the sensitivity was lower than with PEG-precipitated IgG from 0.2 ml serum. The clinical usefulness of the sensitive TSAb assay using PEG-precipitated IgG or direct serum assay in the presence of high PEG concentrations was demonstrated.     

Relationship among thyrotropin (TSH), thyroid stimulating immunoglobulins, and results of triiodothyronine (T3) suppression test in patients with Graves' disease

To investigate the relationship between TSH and abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease in whom normal thyroid hormone levels in the serum were maintained by antithyroid drug therapy and in patients with euthyroid Graves' disease, determinations were made of the TSH concentration, action of thyroid stimulating immunoglobulins (TSAb and TBII), and T3 suppression. Out of thirty-three patients with hyperthyroid Graves' disease, twelve patients with subnormal TSH levels were all non-suppressible according to the T3 suppression test results and the detectability of TSAb and/or TBII was as high as 75%. In three out of five patients with euthyroid Graves' disease, the serum TSH level was subnormal. All three showed non-suppressibility in the T3 suppression test and positive action of either TSAb or TBII. One of them became clinically thyrotoxic when the TSAb activity was further increased and TBII became positive, and was therefore diagnosed as having hyperthyroid Graves' disease. The present findings suggest that there are still abnormal thyroid stimulator(s) in patients with hyperthyroid Graves' disease who have low TSH, even if their thyroid hormone concentrations remain normal. Moreover, it is likely that some of the patients with euthyroid Graves' disease are actually in a state of subclinical hyperthyroidism because of the presence of abnormal thyroid stimulator(s).     

Stimulation of adenyl cyclase and fixation of iodine in human cancerous thyroid cells in culture

In primary cultures of cells from human thyroid cancer, functional activity was investigated by adenylate cyclase responsiveness and radioiodine uptake. In cells from well-differentiated follicular carcinomas (2 cases), TSH stimulation of cAMP accumulation is similar to that of normal thyroid cells. In contrast, in papillary carcinomas (11 cases), the mean dose-response curve is much lower than normal. In thyroid cancers, radioiodine uptake by the cells has been detected in some cases after 3 days of 125I incorporation. As previously observed, our results suggest the existence of a relationship between adenylcyclase responsiveness and 125I uptake by the cells.     

Identification of separate determinants on the thyrotropin receptor reactive with Graves' thyroid-stimulating antibodies and with thyroid-stimulating blocking antibodies in idiopathic myxedema: these determinants have no homologous sequence on gonadotropin receptors.

Deletions, substitutions, or mutations of the rat TSH receptor extracellular domain between residues 20 and 107 (all residue numbers are determined by counting from the methionine start site) have been made by site-directed mutagenesis of receptor cDNA. After transfection in Cos-7 cells, constructs were evaluated for their ability to bind [125I]TSH or respond to TSH and thyroid-stimulating antibodies (TSAbs) from Graves' patients in assays measuring cAMP levels of the transfected cells. Assay results were compared to results from Cos-7 cells transfected with wild-type receptor constructs or vector alone. We identify threonine-40 as a TSAb-specific site whose mutation to asparagine, but not alanine, reduces TSAb activity 10-fold, but only minimally affects TSH-increased cAMP levels. We show that thyroid-stimulating blocking antibodies (TSBAbs), which block TSH or TSAb activity and are found in hypothyroid patients with idiopathic myxedema, continue to inhibit TSH-stimulated cAMP levels when threonine-40 is mutated to asparagine or alanine, suggesting that TSBAbs interact with different TSH receptor epitopes than the TSAb autoantibodies in Graves' patients. This is confirmed by the demonstration that these TSBAbs interact with high affinity TSH-binding sites previously identified at tyrosine-385 or at residues 295-306 of the extracellular domain of the TSH receptor. This is evidenced by a loss in the ability of TSBAbs to inhibit TSAb activity when these residues are mutated or deleted, respectively. Since the TSAb and TSBAb epitopes are in regions of the extracellular domain of the TSH receptor that have no homology in gonadotropin receptors, these data explain at least in part the organ-specific nature of TSH receptor autoantibodies in autoimmune thyroid disease. Data are additionally provided which indicate that residues 30-37 and 42-45, which flank the TSAb epitope at threonine-40, appear to be ligand interaction sites more important for high affinity TSH binding than for the ability of TSH to increase cAMP levels and that cysteine-41 is critical for TSH receptor conformation and expression on the surface of the cell. Thus, despite unchanged maximal values for TSH-increased cAMP levels, substitution of residues 42-45 or deletion of residues 30-37 results in receptors, which, by comparison to wild-type constructs, exhibit significantly worsened Kd values for TSH binding than EC50 values for TSH- or TSAb-increased cAMP activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Augmentative effect of polyethylene glycol, polyvinyl alcohol and dextran on thyroid-stimulating antibody stimulated cAMP production in FRTL-5 cells and CHO cells expressing human TSH receptor

Previously we reported the augmentative effect of nonionic hydrophilic polymers such as polyethylene glycol (PEG), polyvinyl alcohol (PVA) and dextran on thyroid-stimulating antibody (TSAb) activity in porcine thyroid cell assays. We examined whether a similar phenomenon occurs in FRTL-5 thyroid cells and CHO cells expressing the human (h) TSH receptor (CHO-hTSHR cells). As with porcine thyroid cells, PEG 22.5% precipitated crude IgG from serum of patients with Graves' disease, significantly increased cAMP production as compared with PEG 12.5% precipitated crude IgG in both FRTL-5 cells and CHO-hTSHR cells. PEG 5% augmented purified-TSAb-IgG-stimulated cAMP production in both cell assays. TSAb activities and positivity by the direct assay using whole serum (0.05 ml) in the presence of 5% PEG in untreated Graves' patients were significantly increased as compared with the absence of 5% PEG. The augmentative effects of PVA 10% or dextran T-70 10% on TSAb-IgG-stimulated cAMP production were also observed in both cell assays. PVA 10% did not augment TSH-stimulated cAMP production in spite of weak augmentation by dextran 10% in both cell assays. Lack of the augmentative effects of PEG 5%, PVA 10% and dextran 10% on cAMP produced by GTPgammaS, forskolin and pituitary adenylate cyclase activating polypeptide was observed in both cell assays. The augmentative effects of these polymers in both cell assays similar to porcine thyroid cells suggest that there is no apparent species specificity among human, porcine and rat thyroid cells as far as TSH receptor linked cAMP production in cell membranes existed.     

Hormonal regulation of thyroid peroxidase in normal and transformed rat thyroid cells

The hormonal induction of thyroid peroxidase (TPO) mRNA is studied in the functional rat thyroid cell line FRTL-5 and compared to the induction of thyroglobulin (TG) mRNA and I- uptake. TPO and TG mRNAs are regulated by TSH and by insulin-like growth factor I (IGF-I) and/or insulin. However, while TPO is more sensitive to TSH regulation (5- to 6-fold increase vs. 2- to 3-fold increase by IGF-I), TSH and IGF-I are equally potent in increasing TG mRNA levels (3- to 4-fold). Regulation of I- uptake appears to be different: thus TSH greatly (15-fold) increases I- uptake, while IGF-I or insulin are completely ineffective. TPO and TG mRNAs and I- transport display different sensitivity to transformation of rat thyroid cells. Thus, when another differentiated rat thyroid cell line, the PC cells, are transformed by human c-myc (PC myc), TPO and TG mRNAs are both present at normal levels, while I- uptake is slightly decreased; in the PC cells transformed by polyomavirus middle-T-antigen (PC PyMLV) TPO mRNA is undetectable and I- uptake is greatly decreased, while TG mRNA is present at normal levels. All three differentiated functions are switched off in PC cells transformed by the cooperation of c-myc and polyomavirus middle-T-antigen (PC myc + PyMLV).     

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.