Thyroid-stimulating activity of human chorionic gonadotropin in sera of normal pregnant women.

To ascertain the thyrotropic activity of human chorionic gonadotropin in sera of normal pregnant women, we examined the adenylate cyclase activation in the cultured FRTL-5 cells by extracted hCG from 7 normal pregnant women. hCG was extracted from the sera using anti-hCG-beta subunit monoclonal antibody-coated microwells, eluted with 2 mol/l guanidine-HCl, and reconstituted with hypotonic Hanks' solution. FRTL-5 cells were precultured in 5H medium, incubated for 2 h with the serum extracts, and the cAMP released into the medium was measured. hCG levels in serum extracts ranged from 1100 to 6800 IU/l; values corresponded to 1.4-19.8% compared with those in the original serum samples. Addition of the extracts to FRTL-5 cells resulted in significant increases in the cAMP accumulation, ranging from 9.8 to 59.0 nmol/l. cAMP levels were also increased in a dose-dependent manner by adding purified hCG as well as crude hCG and hTSH to FRTL-5 cells. These findings suggest that the thyroid gland of normal pregnant women may actually be stimulated by hCG itself.     

Human chorionic gonadotropin stimulates iodide uptake, adenylate cyclase, and deoxyribonucleic acid synthesis in cultured rat thyroid cells

hCG stimulates thyroid function, but it has been suggested that it is impurities in commercial hCG preparations or a variant of hCG that are responsible for the thyrotropic activity. In this study, we tested the thyrotropic activity of purified and commercial hCG and compared its action with that of bovine TSH (bTSH) in cultured rat FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and synthesis of DNA. Iodide uptake was measured after incubation of the cells for 48-72 h with the test hormones, followed by a 40-min incubation with 0.1 microCi Na125I and 10 mumol/L carrier NaI; the 125I in the washed cells was counted. Adenylate cyclase was measured after incubation of the cells with the test stimulators for 3 h in hypotonic medium by RIA of cAMP in the medium. DNA synthesis was measured after incubation of the cells with the test substances for 24 h, followed by addition of [3H]thymidine for 3 h and then measuring the incorporation of [3H]thymidine into the cells. Both purified and commercial hCG produced a dose-related increase in iodide uptake. The relative potency of commercial hCG was 0.024 microU bTSH/U hCG and that of purified hCG was 0.042 microU bTSH/U hCG; compared with human TSH, the potency of purified hCG was 0.72 microU/U hCG. hCG caused a dose-related increment of adenylate cyclase and [3H]thymidine incorporation. The effect of hCG on iodide uptake and [3H]thymidine incorporation was additive with that of bTSH; hCG was not an antagonist of TSH in these cultured rat thyroid cells. We conclude that hCG has intrinsic thyrotropic activity in FRTL-5 cells in regard to stimulation of iodide uptake, activation of adenylate cyclase, and stimulation of DNA synthesis.     

STIMULATION OF CYCLIC AMP PRODUCTION IN FRTL-5 THYROID CELLS BY CRUDE IMMUNOGLOBULIN FRACTIONS OF SERUM FROM PREGNANT WOMEN

In an assay for thyroid-stimulating antibodies, in which FRTL-5 thyroid cells were incubated with crude immunoglobulin (Ig) fractions precipitated from serum with 15% polyethylene glycol, significant increase in cAMP production was elicited by the samples from 25 (35.7%) out of 70 pregnant women. The highest value was 529.5%. There was a close correlation between thyroid stimulating activities and serum hCG concentrations (r = 0.708, P less than 0.001). When 125I-hCG was added to serum from pregnant women, about 20% of the radioactivity was incorporated into the Ig fractions. hCG preparations within a range of concentrations of 30-300 IU/m elicited 2.3-16.5 times increase in cAMP in a dose-dependent manner. Nine pregnant women with serum TSH concentrations less than the lower limit of the normal range (less than 0.25 mU/l) displayed significantly higher values for both thyroid stimulating activities and serum hCG concentrations (P less than 0.001, respectively) compared with those who had normal TSH levels in serum. These data suggest that hCG or its variant may stimulate the thyroid sufficiently to suppress secretion of TSH from the pituitary in some pregnant women.     

Thyroid-stimulating activity in sera of normal pregnant women

To evaluate the effect of a thyroid stimulator on thyroid function in the sera of normal pregnant women, we measured thyroid-stimulating activity (TSA) using a highly sensitive bioassay based on cAMP accumulation in cultured rat FRTL-5 thyroid cells. Serum was pretreated with 10% polyethylene glycol (PEG), and the supernatant (PEG-pretreated serum) was then used in the following studies. FRTL-5 cells were preincubated in 5H medium and incubated for 2 h with PEG pretreated serum, and cAMP was measured. All 11 patients with untreated hyperthyroid Graves' disease with strongly positive thyroid-stimulating antibody activity had normal TSA, because only 5.6% of their immunoglobulin G was recovered in the PEG-pretreated serum. In 32 normal pregnant women, 29 (91%) had positive TSA. Their TSA showed statistically significant positive correlations with serum hCG and free T4 levels, and a negative correlation with serum TSH levels. Moreover, when hCG was absorbed from sera by incubation with the solid phase anti-HCG monoclonal antibody, a significant positive correlation was observed between the rate of decrease in hCG and that in TSA. In conclusion, 1) TSA exists in the sera of normal pregnant women, which reflects hCG itself; and 2) thyroid glands of normal pregnant women may be stimulated by TSA to induce a slight suppression of TSH but not sufficient to induce overt hyperthyroidism.     

Carbohydrate modifications transform human chorionic gonadotropin into a potent stimulator of adenosine 3',5'-monophosphate and growth responses in FRTL-5 thyroid cells.

To delineate the role of carbohydrate moiety in the expression of in vitro thyrotropic activity of hCG, its variants that lacked sialic acid residues or the entire carbohydrate moiety on one or both subunits were prepared. They along with intact hCG were then tested for the abilities to bind to TSH receptor and stimulate cAMP production and growth responses in FRTL-5 cells. The removal of sialic acid from either one or both subunits sharply increased the [125I]bovine TSH binding-inhibiting activity of hCG in the receptor assay. Among the variants tested, desialylated hCG (as-hCG) was the most potent inhibitor, followed by alpha-as-beta, as-alpha-beta, and hCG in that order. With respect to their abilities to stimulate cAMP generation in the cells, the activities of all desialylated hCG variants were markedly higher than that of hCG itself, and as in the receptor assay, as-hCG was the most potent stimulator tested. At the same concentration (100 micrograms/ml), as-alpha-beta, alpha-as-beta, and hCG were approximately only 57%, 46%, and 27% as active as as-hCG in activating cAMP production. The findings in the growth assay were entirely consistent with those noted in the cAMP response assay. Among the deglycosylated hybrids examined, alpha-dg-beta was the most effective in activating cAMP release. An equivalent dose (200 micrograms/ml) was about 1.7, 2.7, and 3.8 times as active as dg-hCG dg-alpha-beta, and hCG, respectively. The deglycosylated variants of hCG showed a similar pattern of activity in growth response and cAMP accumulation assays. In both cases, alpha-dg-beta was the most potent stimulator, while hCG was the least active. No significant difference between the potencies of dg-alpha-beta and alpha-dg-beta was discerned in the receptor assay; however, deglycosylated hCG (dg-hCG) was sharply more active. Results of these studies strongly suggest that the optimal expression of in vitro thyrotropic activity of hCG variants in FRTL-5 cells may require an alpha-subunit, with intact carbohydrate, in combination with the deglycosylated beta-subunit. Further, they demonstrate that modification of the hCG carbohydrate moiety alone can transform it from a weak agonist to a potent stimulator of in vitro thyrotropic bioactivity. These results also provide further evidence to support the notion that the degree of expression of thyrotropic activity is strongly affected by the species in which the studies are performed.     

Binding of bovine thyrotropin to receptors in rat testis and its interaction with gonadotropins.

Previously, we have shown that preparations of hCG bind to bovine thyroid membranes, as judged from their ability both to inhibit the binding of 125I-labeled bovine TSH (bTSH) and to activate adenylate cyclase (Amir, S.M., H. Uchimura, and S.H. Ingbar, J Clin Endocrinol Metab 45: 280, 1977). In the present studies, 125I-labeled, highly purified bTSH ([125I]bTSH) has been shown to bind specifically and saturably to receptors in a particulate fraction from rat testis. At 37 C, binding was rapid, reaching a maximum level in less than 15 min, but then declining markedly during the next several hours. At 22 C, binding reached a steady state after 2 h and remained unchanged for another 22 h. Binding of [125I]bTSH was greatest at pH 5.5, at which pH more than 50% of [125I]bTSH was bound in the presence of 330 microgram/ml particulate protein, the concentration of protein that yielded maximum binding. Nevertheless, the majority of experiments were conducted at lesser protein concentrations and at physiological pH (7.45), under which conditions total binding was only 25% of that measured at pH 5.5. Scatchard plots indicated the presence of a single binding site with a dissociation constant of 5.8 X 10(-8) M and a binding capacity of 0.22 nmol/mg protein on the basis of data obtained at 22 C and pH 7.45. Both crude and highly purified preparations of hCG inhibited the binding of [125I]bTSH to testis particulate fraction; crude hCG had 46 times the activity, and purified hCG had only one-tenth the activity of bTSH itself in this respect. This was true despite the fact that with respect to the displacement of [125I]hCG, crude and purified hCG were almost equally active. Bovine LH had one-third the activity of bTSH in displacing [125I]bTSH. Human FSH inhibited [125I]bTSH binding only slightly at the highest concentration tested, while glucagon, insulin, PRL, and GH were inactive. Purified bTSH inhibited the binding of [125I]hCG to testis particulate fraction but contained only about 2% of the activity of purified hCG. Lineweaver-Burk analysis suggested that inhibition of [125I]hCG binding by bTSH was competitive in nature. Purified bTSH stimulated cAMP production in Leydig cells, but with only about 0.1% of the activity of purified hCG. It is concluded that bTSH binds reversibly, saturably, and with relatively high affinity to receptors in rat testis that are either the same as receptors for hCG and LH or that interact therewith. bTSH, like hCG, is capable of stimulating the production of cAMP in rat Leydig cells, but is much less potent than hCG in this regard. Preparations of crude hCG contain a factor lacking hCG activity in bioassay, immunoassay, and receptor assay that is especially potent in displacing [125I]bTSH from receptors in testis, as has earlier been described for bTSH receptors in bovine thyroid membranes.     

THYROID FUNCTION IN CHORIOCARCINOMA: DEMONSTRATION OF A THYROID STIMULATING ACTIVITY IN SERUM USING FRTL-5 and HUMAN THYROID CELLS

Hyperthyroidism is a well recognized complication of gestational trophoblastic tumours (GTT) and may be due to high circulating concentrations of human chorionic gonadotrophin (hCG) or its variants. We have studied 24 clinically euthyroid women with GTT. Eight were biochemically hyperthyroid with low or undetectable serum thyrotrophin (TSH) and had a mean serum hCG of 361.2 x 10(3) IU/l compared to 76.2 x 10(3) IU/l in the other patients (P less than 0.01). Purified hCG stimulated iodide uptake into FRTL-5 cells with 25 x 10(3) IU/l being equivalent in potency to 1 mU/l of thyrotrophin (TSH). Sixteen out of the 24 sera (67%) stimulated iodide uptake when applied to the cells at a 1:10 dilution. Sera from all eight hyperthyroid patients contained thyroid stimulating activity. The mean hCG concentration in the 16 stimulatory sera was 238.2 x 10(3) IU/l compared to 37.1 x 10(3) IU/l in the other eight sera (P less than 0.01). Six men with hCG-secreting testicular tumours were biochemically euthyroid although three of their sera stimulated iodide uptake into FRTL-5 cells. In human thyroid cells the mean cAMP production over 4 h with sera from five healthy controls was 54.2 +/- 1.81 pmol/mg cell protein compared to 67.0 +/- 3.8 pmol/mg protein with sera from five choriocarcinoma patients (P less than 0.02). Serum from patients with gestational trophoblastic tumours contains a thyroid stimulating activity which may be hCG and whose presence correlates with hyperthyroidism.     

Thyrotropic activity of crude hCG in FRTL-5 rat thyroid cells

The presence of thyroid stimulating activity in partially purified hCG was investigated using, as bioassay system, iodide uptake in rat thyroid FRTL-5 cells. The biological responses evoked by hCG were tested after neutralisation with monoclonal and polyclonal antisera to hTSH and hCG, and after fractionation on Sephadex G-100. The molar amounts of TSH and hCG in respective preparations were calculated assuming an activity of 30 IU/mg and 19 IU/mg, respectively, for bTSH and hTSH, and of 14,000 IU/mg for hCG. A dose-dependent response, paralleling that evoked by bTSH, was observed in a concentration range of 0.1-4 mumol/l hCG; 1 mumol of hCG was equivalent to 50 pmol of bTSH and 132 pmol of hTSH. The thyrotropic activity coeluted with hCG immunoactivity on Sephadex G-100. Incubation with monoclonal anti-hTSH antibodies did not affect the stimulatory ability of hCG preparation, indicating that it was not due to hTSH contamination. Similarly, a pretreatment with monoclonal and polyclonal anti-hCG antibodies did not significantly alter the iodide uptake response induced by hCG. These results indicate that the thyrotropic activity in partially purified hCG is not due to the presence of aspecific contaminants, but to a substance structurally related to hCG in terms of molecular weight. However, it appeared to differ from hCG immunologically, suggesting the hypothesis that minor modifications in the molecular structure may confer thyrotropic activity on hCG, altering its immunoreactive potency.     

Interaction of human chorionic gonadotropin and human luteinizing hormone with human thyroid membranes

In an attempt to identify a possible pathogenetic role for the hCG molecule in the mechanism of the hyperthyroidism which occurs in choriocarcinoma, we have looked for evidence that the hCG molecule has a thyrotropic action on the human thyroid. The thyrotropic activity of various hCG preparations on the human thyroid was assessed by measuring the stimulation of adenylate cyclase activity in human thyroid plasma membranes purified by sucrose density gradient centrifugation. The highly purified hCG CR119 preparation stimulated human thyroid adenylate cyclase activity. Its activity was more than 654 times greater than could be accounted for by human TSH (hTSH) contamination of the preparation, as determined by RIA. The thyrotropic activity intrinsic to 1.0 IU hCG was equivalent to roughly 0.27 microU hTSH. Significant saturable binding of the 125I-labeled highly purified hCG preparation to human thyroid membranes was demonstrated, and the bound component was characterized. Its apparent molecular size, subunit composition, and testis receptor-binding characteristics were those of the hCG molecule. Examination of a crude urinary hCG preparation in adenylate cyclase and TSH radioligand assays using human thyroid membranes showed no evidence of any molecule other than hCG with a thyrotropic action on the human thyroid. Given that hCG binds to and stimulates adenylate cyclase activity in human thyroid tissue, as the above data indicate, then human LH (hLH) would be expected to do the same, since hLH and hCG have such strong structural and functional similarities. As anticipated, a highly purified hLH preparation exhibited TSH binding inhibition and adenylate cyclase stimulation. Its activity was more than 1030 times greater than could be accounted for by hTSH contamination of the preparation. The thyrotropic activity intrinsic to 1.0 IU hLH was equivalent to roughly 44 microU hTSH. Thus, in addition to other shared properties, the hLH molecule and the hCG molecule share the ability to interact with human thyroid tissue. These results strongly indicate that the hCG molecule has a thyrotropic action on the human thyroid and support the hypothesis that hCG is the thyrotropic factor that mediates the hyperthyroidism which occurs in patients with hCG-secreting neoplasms.     

Simultaneous measurement of TSH-binding inhibitor immunoglobulin and thyroid stimulating autoantibody activities using cultured FRTL-5 cells in patients with untreated Graves' disease

A new and practical assay was developed using cultured FRTL-5 cells for simultaneous assessment of TS-ab and TSH-binding inhibitor immunoglobulin, allowing direct comparison of these two activities under the same conditions. Subsequent to the TS-ab assay in which extracellular cAMP concentration in Hanks' medium without NaCl was determined, [125I]b TSH in this medium was added to observe the ability of serum Ig to inhibit the binding of [125I]b TSH to FRTL-5 cells. We found a much higher specific binding of [125I]b TSH to FRTL-5 cells and a much greater inhibition of [125I]bTSH binding to the cells exposed to Graves' Ig in hypotonic NaCl-free than in NaCl containing Hanks' medium, indicating that the binding of both TSH and Graves' Ig to the TSH receptor was salt-sensitive. The inhibitory activity of [125I]bTSH binding to the cells was 0.2 +/- 4.6% (mean +/- SD) in 45 normals. Inter-assay coefficients of variation in two positive controls with the mean values of 18.0 and 65.8% were 15.8 and 16.5%, respectively. Among 46 patients with untreated hyperthyroidism owing to Graves' disease, 45 (97.8%) were positive for TS-ab; 35 (76.1%) and 40 (87.0%) were positive for TSH-binding inhibitor in Ig assays using FRTL-5 cells and solubilized porcine thyroid membranes, respectively. TS-ab activities correlated less closely with TSH-binding inhibitory activities determined using FRTL-5 cells (r = 0.576, p less than 0.001) than with those determined using porcine thyroid membranes (r = 0.745, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyrotropic activity of acidic isoelectric variants of human chorionic gonadotropin from trophoblastic tumors

Previous studies have indicated that hCG has a weak intrinsic thyroid-stimulating activity. Differences in the molecular composition and biological activity of hCG in patients with trophoblastic diseases and pregnant women occur, but are not well defined. Therefore, we have studied the effect of serum samples and purified hCG preparations from patients with trophoblastic diseases on T3 release from human and porcine thyroid slices in vitro. We examined 30 serum samples from 13 patients with nonseminomatous testicular germ cell tumors, 3 from women with choriocarcinoma, and 5 from patients with hydatidiform moles. In all but 1 serum sample from the tumor patients, but in none of 11 serum samples of pregnant women, T3-releasing activity was found. Two patients with testicular cancer and 1 patient with molar pregnancy experienced episodes of frank hyperthyroidism. Isoelectric focusing on polyacrylamide gels of tumor sera (n = 15) revealed substantial amounts of acidic isoelectric variants, pI 3.3-3.9, which were only barely detectable in pregnancy sera. The percentage of acidic hCG variants with pI 3.3-4.0 to total hCG with pI 3.3-5.2, as determined by hCG (+hCG beta) RIA of the eluted fractions of polyacrylamide gel isoelectric focussing, varied from 12-45% in sera of tumor patients and from 0-4% in pregnant sera. We purified the acidic variants of hCG with pI 3.6-3.8 (hCGav) from the urine of our patients. The beta-subunit of purified hCGav had a slightly higher mol wt (35,750) than that of hCG CR 119 (34,190) on polyacrylamide gel electrophoresis. The hCGav showed a dose-dependant stimulation of T3 release and cAMP generation from human thyroid slices, whereas the other hCG fractions on isoelectric focussing had no thyrotropic effect in similar dose levels. The TSH-like activity of hCGav could be roughly estimated as 10 mIU TSH/IU hCGav. Anti-hCG (+hCG beta) antiserum, but not anti-hTSH antiserum, neutralized the biological activity of hCGav. These findings strongly suggest that acidic hCG variants act as functional stimulators of the human thyroid in vitro. Since these molecular variants of hCG can exist in patients with trophoblastic diseases in significant amounts, they could be responsible for some cases of hyperthyroidism in trophoblastic diseases.     

The mechanism of action of tumour necrosis factor-alpha and interleukin 1 on FRTL-5 rat thyroid cells

Previous work showed that treatment of rats with tumour necrosis factor-alpha produced a model of nonthyroid illness in which there was reduction of circulating thyroid hormones and TSH, reduced thyroid response to TSH, and reduced thyroid iodide uptake. In vitro studies showed that tumour necrosis factor-alpha binds to a specific receptor on FRTL-5 rat thyroid cells, that TSH increases the number of tumour necrosis factor-alpha receptors, and that tumour necrosis factor-alpha inhibits iodide uptake by these cells. In the present study, we obtained additional data on the effects of tumour necrosis factor-alpha on FRTL-5 cells and studied the mechanism of action of tumour necrosis factor-alpha in these cells. Tumour necrosis factor-alpha inhibited both basal and TSH-stimulated [125I]iodide uptake: tumour necrosis factor-alpha slowed the recovery of [125I]iodide trapping after the cells were exposed to TSH and augmented the loss of the [125I]iodide trapping function after the cells were deprived of TSH: tumour necrosis factor-alpha inhibited [125I]iodide trapping in a noncompetitive manner; tumour necrosis factor-alpha did not affect cell growth of FRTL-5 cells. Interleukin-1 (IL-1) also inhibited basal and TSH-stimulated [125I]iodide uptake, but it stimulated cell growth. Tumour necrosis factor-alpha and IL-1 did not affect the generation of cAMP in the presence or absence of TSH; these cytokines blocked the cAMP-induced stimulation of [125I]iodide uptake. Tumour necrosis factor-alpha did not affect [3H]arachidonic acid uptake or release by FRTL-5 cells. The inhibitors of the phospholipase A2-arachidonic acid pathway did not affect the action of tumour necrosis factor-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measurement of TSAb directly in serum using FRTL-5 cells

FRTL-5 cells were shown to be suitable for the measurement of thyroid stimulating antibody (TSAb) present in sera of patients with Graves' disease. Current methods for the assay of TSAb require the separation of immunoglobulin G (IgG) from patient sera. In this report the possibility to measure TSAb directly on serum was evaluated using FRTL-5 cells. To this purpose cells were seeded in 96-well plates and cultured for 4 days in medium deprived of TSH. Using this system bovine TSH was able to produce a significant stimulation of cAMP production at 1 microU/ml. Whole normal serum completely inhibited the stimulation of TSH as well as that of TSAb, while diluted serum was devoid of any effect. Heat inactivated sera and IgGs, prepared by DEAE Sephadex separation, were diluted in hypotonic medium and incubated with cells for 1 h at 37 C. After incubation cAMP was measured in the assay medium by RIA. In some experiments the effects of graded dilutions of sera and IgGs with known TSAb activity were compared. Sera as well as IgGs increased the cAMP production, but, at the highest concentrations, an inhibitory effect was evident. For this reason sera were tested after appropriate dilution. Thirteen/27 (48%) sera and 22/27 (81%) IgGs from patients with Graves' disease were TSAb positive. The effect of Graves' sera on adenylate cyclase stimulation was completely inhibited by an anti-human IgG. The results of stimulation produced by Graves' sera and IgGs were highly correlated (r = 0.97, p less than 0.001). In conclusion it is possible to measure TSAb directly in serum using FRTL-5 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human chorionic gonadotropin and the thyroid: hyperemesis gravidarum and trophoblastic tumors.

There is abundant evidence that human chorionic gonadotropin (hCG) is a weak thyrotropin (TSH) agonist. In FRTL-5 rat thyroid cells, hCG increases cyclic adenosine monophosphate (cAMP), iodide transport, and cell growth. hCG has thyroid-stimulating activity in bioassays in mice and in clinical studies in man. In cultured cells transfected with the human TSH receptor, hCG increases generation of cAMP. Molecular variants of hCG with increased thyrotropic potency include basic molecules with reduced sialic acid content, truncated molecules lacking the C-terminal tail, or molecules in which the 47-48 peptide bond in the beta-subunit loop is nicked. In normal pregnancy, when hCG levels are highest at 10 to 12 weeks gestation, there is suppression of serum TSH levels, presumably due to slight increases in free thyroxine (T4) concentration. In twin pregnancies, hCG levels tend to be higher and suppressed TSH levels are more frequent. Hyperemesis gravidarum, defined as severe vomiting in early pregnancy that causes 5% weight loss and ketonuria, is usually associated with increased hCG concentration. A high proportion of patients with hyperemesis gravidarum, about one-third to two-thirds in different series, have evidence of increased thyroid function. Only a small proportion of these patients have clinical hyperthyroidism, termed gestational thyrotoxicosis. These patients probably secrete a variant of hCG with increased thyroid-stimulating activity. Trophoblastic tumors, hydatidiform mole, and choriocarcinoma often cause hyperthyroidism because they secrete very large amounts of hCG. When the serum hCG exceeds about 200 IU/mL, hyperthyroidism is likely to be found. There is a correlation between the biochemical severity of hyperthyroidism and the serum hCG in these patients. Removal of the mole or effective chemotherapy of the choriocarcinoma cures the hyperthyroidism. In conclusion, hCG has thyroid-stimulating activity that influences thyroid function early in pregnancy when hCG levels are high. Excessive hCG secretion may cause hyperthyroidism in patients with hyperemesis gravidarum or trophoblastic tumors.     

Thyroid-stimulating activity of chorionic gonadotropin and luteinizing hormone.

While recent evidence indicates that the hCG molecule has intrinsic thyroid-stimulating activity (TSA), it is not clear whether a thyrotropic molecule other than hCG accounts for some of the TSA apparent in the crude or highly purified hCG. To determine if a thyrotropic factor is excluded from crude urinary hCG during purification of hCG, the ratio of interstitial cell-stimulating activity (ICSA) to TSA was determined in the starting material used for hCG preparation as well as in the highly purified hCG preparation. The ratio of the two biological activities did not change significantly during purification, suggesting that no factor present in crude hCG other than hCG itself accounts for the TSA. The highly purified hCG preparation was gel-filtered on Sephadex G-100 and the main protein peak was divided into three fractions. The ratio of TSA to ICSA was the same in each fraction, further indicating that these activities are intrinsic to the same molecule. If hCG has intrinsic thyrotropic activity, as these data indicate, then thyrotropic activity would be expected to be a secondary biological activity of LH, since there are strong structural and functional similarities between LH and hCG. In order to assess the LH molecule for intrinsic TSA, an LH preparation with minimal TSH contamination was prepared by recombining subunits exhibiting minimal TSH immunoreactivity. The LH molecule formed from the recombination of highly purified hCG alpha and ovine LH beta subunits exhibited TSA in the bioassay that was 25 times greater than that expected based on the immunoreactive TSH contamination. There was no evidence to support the existance of a thyrotropic factor other than hCG in either crude or highly purified hCG preparations. Our finding that a hybrid LH molecule structurally similar to hCG with potent ICSA also exhibits intrinsic TSA further extends and supports the hypothesis that TSA is an intrinsic property of the hCG molecule.     

hCG-induced TSH receptor activation and growth acceleration in FRTL-5 thyroid cells

Our previous studies have indicated specificity cross-over between LH/hCG and the TSH receptor. We have now analyzed the ability of the TSH-dependent Fisher Rat thyroid cell line (FRTL-5) to proliferate in a differentiated state under the influence of highly purified hCG (hCG-CR121). TSH receptor activation and growth induction were observed after 7 days suspension from a TSH-induced growth phase. The effect of 10 ug hCG-CR121 was equivalent to 500 +/- 43 (mean +/- SEM) uIU of human TSH (2nd IRP, 80/558) with reference to growth stimulation, as judged by 72-h [3H]-thymidine uptake and to approximately 15 +/- 35 uIU human TSH with reference to receptor activation as judged by cyclic AMP accumulation. These data demonstrate specificity cross-over by hCG for the TSH-dependent FRTL-5 cell line and suggest that hCG has greater growth-stimulating than thyroid-stimulating potential when compared with human TSH.     

Alpha 2- and beta-adrenergic receptors and adenosine A1 receptor of FRTL-5 rat thyroid cells in relation to fucosyl GM1 ganglioside

We have previously reported that anti-fucosyl GM1 ganglioside antibody partially suppresses cAMP production in FRTL-5 rat thyroid cells not via the TSH receptor but via guanine nucleotide-binding protein indirectly. In order to clarify further the mechanism of the antibody action, we studied the relationship with alpha 2- and beta-adrenergic and adenosine A1 receptors. FRTL-5 cells did not bind [3H]clonidine, suggesting the lack of alpha 2-adrenergic receptor or at least abnormality of its binding domain. On the other hand, the cells specifically bound [125I]iodocyanopindolol, but isoproterenol failed to affect the basal and TSH-stimulated cAMP production indicating the lack of coupling with adenylate cyclase. The inhibition of cAMP production induced by anti-fucosyl GM1 antibody was not altered by adrenergic agents. [125I]hydroxyphenylisopropyl adenosine binding was observed in FRTL-5 cells but was not displaced by the antibody. These results lead to conclusions that FRTL-5 cells lack alpha 2-adrenergic receptor but have beta-adrenergic receptor which lacks coupling with adenylate cyclase and have adenosine A1 receptor, and that the adrenergic receptors and adenosine A1 receptor are not the site of action of anti-fucosyl GM1 antibody.     

Divergent responses by human and mouse thyroids to human chorionic gonadotropin in vitro

hCG is a known stimulator of mouse thyroid in vivo. Studies were therefore performed to ascertain whether the thyroid-stimulating activity of hCG in the mouse could also be demonstrated by the in vitro techniques that had failed to show any activity of hCG in the human thyroid. When labeled with 125I and incubated at 22 degrees C in 20 mM Tris-0.5% bovine serum albumin (Tris-BSA), pH 7.45, with increasing concentrations (70-300 micrograms protein/ml) of a mouse thyroid fraction, a purified hCG preparation [( 125I]hCG) showed 5-12% specific binding. In contrast, its binding to a human thyroid particulate fraction, over the same range of protein concentrations, did not exceed 1%. When similar studies were performed at 37 degrees C in 10 mM Tris-50 mM NaCl-0.5% BSA, pH 7.45, [125I]hCG showed no detectable binding either to the human or the mouse thyroid fractions. At concentrations ranging from 1 to 20 mIU/ml (0.9-18 X 10(-9) M), bTSH stimulated cAMP release from human thyroid slices into the medium in a dose-dependent manner. In contrast, hCG concentrations from 10(3) to 10(4) IU/ml (2-20 X 10(-6) M) were without effect on cAMP release. bTSH, at concentrations of 4.5 and 9.0 mIU/ml (4 and 8 X 10(-9)M), stimulated cAMP release from the mouse thyroid, producing in the medium approximately 11- and 28-fold increases in cAMP concentration. hCG also stimulated cAMP release from the mouse thyroid, the increases being approximately 2.3- and 1.8-fold, in the presence of 2270 and 4540 IU/ml (4.5 and 9.0 X 10(-6) M), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Graves' IgG stimulation of iodide uptake in FRTL-5 rat thyroid cells: a clinical assay complementing FRTL-5 assays measuring adenylate cyclase and growth-stimulating antibodies in autoimmune thyroid disease

With optimal conditions and cells maintained in the absence of thyrotropin (TSH) for 7-10 days, IgG preparations from approximately 90% of patients with active Graves' disease can exhibit statistically significant stimulation of cAMP levels in rat FRTL-5 thyroid cells as compared to normal controls. FRTL-5 cells maintained in the absence of TSH for 7-10 days lose their ability to take up iodide. Iodide uptake returns upon readdition of TSH over a 60-hour period via a cAMP-mediated process; thus TSH can be replaced by dibutyryl cAMP or other agents which increase cAMP levels, for example, thyroid-stimulating autoantibodies (TSAbs) from Graves' sera. TSAb stimulation of iodide uptake requires the continued presence of TSAb over at least the first 24 hours of a 48-hour reversal period; TSH, in contrast, can be withdrawn after 5 hours and will still achieve maximal effects at 36-48 hours. Iodide uptake, measured as a 30-minute pulse at 48 hours, appears, however, to be faster with TSAb than TSH. With optimized conditions (cells depleted of TSH greater than 7-10 days; 3-isobytyl-1-methyl xanthine, 0.005 mM; TSAb addition for the entire 48-hour assay period; and a 30-minute pulse of 10 microM 125I-sodium iodide at 37 C), TSAb stimulation is concentration-dependent with a half-maximal activity at approximately 10-fold lower concentrations than in the cAMP stimulation assay. In a series of 24 patients with Graves' disease, IgGs with positive values in the cAMP assay were positive in the iodide uptake assay.(ABSTRACT TRUNCATED AT 250 WORDS)