Thyroid Adenylate Cyclase Research Articles

Familial Clustering of Thyroid Stimulation-Blocking Antibody

Thyroid stimulation—blocking antibody (TSBAb), an antibody that blocks stimulation of thyroid adenylate cyclase induced by thyrotropin stimulation, 1 has been considered to play some pathogenetic role in atrophic autoimmune primary hypothyroidism. 2,3 We describe a familial clustering of TSBAb that affected 4 members of 1 family. We believe that these cases provide a novel mechanism of hereditary hypothyroidism. Report of Cases. The proband, a 63-year-old woman, presented with general malaise and edema. Her goiter was not palpable. She was found to have hypothyroidism (free thyroxine, 4 pmol/L [0.3 ng/dL]; thyrotropin, 149 mU/L) and was positive for both thyrotropin receptor antibody (88.1%) and TSBAb (92.8%). Serologic tests were also positive for antithyroglobulin and antithyroid microsomal antibodies (X200 and X200). Her daughter, who had been diagnosed as having hypothyroidism with TSBAb (73.0%), had been treated with levothyroxine (100 μg/d) for several years. Her son had once been treated in another clinic under

Humoral thyroid autoimmunity is not involved in the pathogenesis of myxedematous endemic cretinism.

A role of thyroid autoimmunity in the pathogenesis of myxedematous endemic cretinism was suggested by reports indicating the presence of thyroid growth-blocking antibodies in the sera of these patients. To check this hypothesis, we searched for TSH receptor antibodies with thyroid growth-blocking or adenylate cyclase (AC)-inhibiting (TSH-blocking) activity in immunoglobulin G (IgG) from 18 euthyroid and 21 hypothyroid endemic cretins living in Italy and Peru. Among hypothyroid cretins, 12 had no palpable goiter. Stages I-III goiters were present in 12 of 18 euthyroid cretins. Controls included 25 euthyroid nongoitrous subjects living in the same endemic regions as cretins, and 10 normal subjects from an iodine-sufficient area. IgG from 4 selected patients with autoimmune atrophic thyroiditis and from 2 neonates with sporadic transient congenital hypothyroidism due to maternal TSH-blocking antibodies were included in the study. The blocking effect of the IgG was assessed in FRTL-5 cells by measuring TSH-stimulated [3H]thymidine incorporation, DNA accumulation, and AC activation. A radioreceptor assay was used to detect TSH-binding inhibiting antibodies (TBIAb). No IgG from hypothyroid endemic cretins without goiter contained TBIAb or inhibited TSH-stimulated cell growth or AC activation. The effect of IgG from hypothyroid nongoitrous cretins did not differ from that produced by IgG from hypothyroid cretins with goiter, euthyroid cretins with or without goiter, or normal controls. In contrast to these results, IgG from patients with autoimmune atrophic thyroiditis and from neonates with sporadic transient congenital hypothyroidism contained TBIAb that inhibited both TSH-stimulated cell growth and AC activation. In conclusion, our results indicate that, similar to other types of endemic cretinism, hypothyroid endemic cretins without goiter do not have TSH receptor antibodies able to inhibit TSH-stimulated thyroid cell growth or function. These observations argue against a role of humoral thyroid autoimmunity in the development of myxedematous endemic cretinism.

Thyroid autoantigens and their relevance in the pathogenesis of thyroid autoimmunity

Humoral and cellular immune responses are both involved in autoimmune disorders of the thyroid gland. In the last five years, new substantial data have been obtained on the nature and the expression of thyroid cell surface autoantigens and on the demonstration of the functional heterogeneity of autoantibodies to the thyroid stimulating hormone (TSH) receptor. In the present report, attention will be mainly focused on recent studies carried out in our laboratory. The main autoantigens so far identified include the ‘microsomal’ antigen, thyroglobulin and the TSH receptor. For many years the ‘microsomal’ antigen (M) was considered a poorly characterized constituent of the cytoplasm of the thyroid cell. In the last five years, several lines of evidence were provided indicating that M is also well represented on the surface of the follicular cell and is identical to thyroid peroxidase (TPO). The use of anti-TPO monoclonal antibodies, presently available, have confirmed this antigenic identity. Microsomal (anti-TPO) antibodies are very useful markers of autoimmune thyroid disorders and are generally present in Hashimoto's thyroiditis, idiopathic myxedema and Graves' disease. TSH receptor antibodies (TRAb) are present in the sera of patients with Graves' disease. TRAb are able to stimulate thyroid adenylate cyclase and also to mimic TSH in its thyroid growth stimulation. Thus, these antibodies may have a pathogenetic role in goiter formation and in thyroid hyperfunction in Graves' disease. TRAb were also shown to inhibit both TSH binding to its receptor and TSH-stimulated adenylate cyclase activity. Recently TRAb, which inhibited TSH-stimulated adenylate cyclase activity, were found in idiopathic myxedema patients and may be responsible for impairment of thyroid function. In conclusion, it appears that the majority of thyroid autoantibodies may interfere in some peculiar thyroid function activities.

Simultaneous assay of thyroid adenylate cyclase- and growth-stimulating antibodies using FRTL-5 cells. Evidence suggesting their identity in patients with Graves' disease.

The relationship between thyroid growth-stimulating antibodies (TGSAb) and thyroid adenylate cyclase-stimulating antibodies (TSAb) in patients with Graves' disease is still a matter of controversy. To investigate this problem, we have developed an assay for the simultaneous measurement of TSAb and TGSAb using FRTL-5 cells. TSAb was detected by its ability to stimulate iodide (I-) uptake and TGSAb by the 3H-thymidine ([3H]-Tdr) incorporation assay. Thirty-four immunoglobulin G (IgG) preparations from patients with active Graves' disease were selected from a previous series in order to include both TSAb-negative IgGs (n = 9) and TSAb-positive IgGs (n = 25) by the cAMP stimulation assay, with a wide range of stimulatory activity. With one exception, the TSAb-positive IgGs produced a significant stimulation of I- uptake; 20 of them were also TGSAb-positive. The nine IgGs negative in the cAMP assay, were also negative in the I-uptake and the [3H]-Tdr incorporation assays. The majority of samples had a similar potency in the two assays and a significant positive correlation was found (r = 0.76; P less than 0.001). Two IgGs previously shown to inhibit TSH-stimulated adenylate cyclase in FRTL-5 cells produced an almost complete inhibition (80-90%) of both TSH- and Graves' IgG-stimulated I- uptake and [3H]-Tdr incorporation. In conclusion, using a simultaneous assay for thyroid growth and adenylate cyclase stimulation, TGSAb in Graves' patients were found only in TSAb-positive IgGs; both Graves' IgG-stimulated activities were inhibited by antibodies blocking the TSH-dependent adenylate cyclase stimulation. Our data strongly suggest that the same antibody may be responsible for both goitre and thyroid hyperfunction of Graves' disease.

Sera of patients with idiopathic myxedema contain IgG which block TSH-stimulated thyroid adenylate cyclase.

Sera of patients with idiopathic myxedema contain IgG which block TSH-stimulated thyroid adenylate cyclase.

Desensitization of the thyroid cyclic AMP response to thyroid stimulating immunoglobulin: Comparison with TSH

Studies were conducted to examine the characteristics of thyroid cell cAMP stimulation by thyroid stimulating immunoglobulins (TSI) and to compare the cAMP response to TSI and TSH in desensitized human thyroid cells. In terms of cAMP production, preexposure (eight hours) of the cells to TSI induced a desensitization very similar to TSH-induced desensitization: (1) both TSH- and TSI-desensitized cells showed a normal response to cholera toxin and forskolin stimulation; (2) TSH and TSI desensitization was interchangeable in that desensitization by either stimulator affected the action of the other; (3) the time of recovery from either TSH and TSH desensitization was identical; (4) the cycloheximide (10 −4 mol/L) prevented both TSI- and TSH-induced desensitization; (5) preexposure of the cells to iodine, which affects mainly the adenylate cyclase catalytic unit, or to epinephrine, which activate the inhibitory regulatory protein Ni by the α 2-adrenergic stimulation, induced a similar inhibition of the subsequent stimulation by both TSH or TSI. The remarkable similarities between TSH and TSI in stimulating and desensitizing thyroid cells strongly support the concept that TSI activates thyroid adenylate cyclase by interacting with the TSH receptor and not through an allosteric mechanism.

Effect of gangliosides on thymic and thyroid adenylate cyclase activity in chicken ontogenesis: [formula omitted], Institute of Biochemistry, Tashkent, USSR

Effect of gangliosides on thymic and thyroid adenylate cyclase activity in chicken ontogenesis: [formula omitted], Institute of Biochemistry, Tashkent, USSR

Stimulation of thyroid adenylate cyclase activity by sera from patients with non-thyroidal illness.

We have previously shown that sera from many hypothyroid patients stimulated adenylate cyclase activity as measured by serum bioactive TSH concentrations produced by FRTL-5 cell line. This TSH-stimulating activity did not correlate with serum immunoreactive TSH. IgG fractions of these sera did not stimulate FRTL-5 cells. The present study was, therefore, undertaken to investigate the thyroid stimulating activity of sera from patients with non-thyroidal illness. Studies were performed in 36 patients with various non-thyroidal illness. In these patients, serum concentrations of T4, free thyroxine, T3, and TSH were determined. In addition, sera were incubated with FRTL-5 cells or porcine thyroid cells in primary culture in the presence of 0.4 mM MIBX, and medium cAMP concentrations were determined by radioimmunoassay. Sera obtained from some patients with various non-thyroidal illness increased cAMP concentrations in culture media of FRTL-5 cells as well as that of porcine thyroid cells. The thyroid stimulating effects of sera were not disease specific and significantly correlated inversely with serum T3 and T4 concentrations. Serum TSH concentrations in these patients were within the normal range even by the newly developed ultrasensitive assay. Although the nature of substance(s) present in sera of patients with low T3 syndrome which stimulates thyroid adenylate cyclase is not entirely known, it is conceivable that there exist mechanisms independent of TSH to compensate the decreased serum T3 levels in low T3 syndrome.

Activation of thyroid adenyl cyclase by antisera to thyroid plasma membrane preparations: Effects of IgG and non-IgG antiserum components

We have previously shown that IgG isolated from rabbit antibovine thyroid plasma membrane (anti-BTPM) antibodies exhibits properties similar to long acting thyroid stimulator (LATS) and HTS-Ig in that it activates thyroid adenyl cyclase. In order to test whether another immunoglobulin class, eg. IgM, of anti-BTPM antiserum can also stimulate the bovine thyroid adenyl cyclase system, protein molecules of the antiserum were separated into different molecular sizes by gel filtration chromatography on Sephadex G-200. It was observed that the low molecular weight fraction, consisting predominantly of albumin, was inactive in stimulating adenyl cyclase of the thyroid gland. In contrast, both IgM-enriched and IgG-enriched fractions of the immune serum were fully active. Furthermore, the thyroid-stimulating activity of the IgM-enriched fraction can only be inhibited by anti-IgM and that of the IgG-enriched fraction by anti-IgG. Our data suggest that IgM, in addition to IgG, may also have LATS or LATS-like activities in terms of activating adenyl cyclase of the thyroid gland.

Studies on monoclonal antibodies to TSH receptors--heterogeneity and pathophysiological significance of antibodies to TSH receptor

Recent studies revealed that anti-TSH receptor autoantibodies are involved in the pathogenesis of both Graves' disease and a part of hypothyroidism, but precise mechanism of action of these antibodies remained to be studied. In order to delineate the heterogeneity of these antibodies and their pathophysiological significance, we produced monoclonal antibodies to TSH receptor and studied their characteristics. Mouse monoclonal antibodies to TSH receptor were derived from spleen cells of mice immunized with partially purified human TSH receptor, which was prepared by TSH-coupled affinity chromatography of thyroid membrane solubilized with Triton X-100. By fusing spleen cells and mouse myeloma cells in the presence of polyethylene glycol and selecting with limiting dilution method, 5 hybridomas were obtained. Among 3 antibodies, which inhibited TSH binding to thyroid membrane (TSH displacing activity, TDA), 2 inhibited TSH stimulation of thyroid adenylate cyclase (AC) (human thyroid adenylate cyclase inhibitor activity, HTACI), and one showed no bioactivity. Among other 2 antibodies without TDA, 1 stimulated AC (human thyroid adenylate cyclase stimulator activity, HTACS) and the other inhibited TSH stimulation (HTACI). All activities of these antibodies were dependent on IgG concentration and disappeared by treatment of anti-mouse IgG antibodies. In addition, 4 human-human hybridomas were established by fusing human peripheral lymphocytes of patients with Graves' disease and nongoitrous hypothyroidism with human lymphoblastoid cell line. Among 2 antibodies with TDA, one antibody inhibited TSH stimulation of AC, inhibiting TSH binding competitively and another antibody stimulated AC, inhibiting TSH binding noncompetitively. Among the other 2 antibodies, which did not inhibit TSH binding but were shown to bind to TSH receptor by immunoprecipitation, one stimulated AC and the other inhibited TSH stimulation of AC. Among 2 antibodies with HTACI, one antibody with positive TDA inhibited stimulation of AC by stimulative antibodies with positive TDA, but the other without TDA inhibited stimulation of AC by both antibodies with or without positive TDA. These inhibitory antibodies did not inhibit stimulation of AC by Forskolin and Gpp(NH)p, which are known to affect other parts of receptor-AC system than receptor unit. These data suggest that anti-TSH receptor antibodies are heterogenous in the mode of binding to the receptor and in their bioactivities, and may be involved in the pathogenesis of both Graves' disease and a part of idiopathic hypothyroidism.

Can the inhibition of prostaglandin synthesis affect the adenylate cyclase response to thyroid stimulating antibodies?

The majority of Graves' immunoglobulins have the capacity to stimulate thyroid adenylate cyclase (AC) activity in vitro. So far, the exact events leading to AC activity stimulation by thyroid stimulating antibody (TSAb) are not known. It has been suggested that TSAb activates thyroid AC through prostaglandin (PG) synthesis, implying the concept that TSH and TSAb stimulate AC in different ways. If this is so, the inhibition of PG synthesis should notably reduce the response of thyroid plasma membrane to TSAb. We have, therefore, investigated the effect of 2 inhibitors of PG synthesis on the TSAb-stimulated AC activity in a crude human thyroid plasma membrane preparation. Neither indomethacin, nor hydrocortisone, even at concentrations able to completely inhibit PG synthesis, had any significant effect on the thyroid plasma membrane response to TSAb. The results suggest, therefore, that, at least under our experimental conditions, PG does not mediate the AC activation by TSAb. Consequently, the aforementioned suggestion should not be used to claim that TSH and TSAb activate thyroid AC through different pathways. The data, however, do not exclude that TSAb may determine PG synthesis in thyroid cells through the phosphatidylinositol signal system (Pl system).

Separation and analysis of two plasma membrane fractions from bovine thyroid which differ in TSH binding and TSH activation of adenylate cyclase

A tissue disruption technique leading to the separation of thyroid epithelial cell components from interfollicular material has been used to study the distribution and the properties of membrane adenylate cyclase originating from intraglandular thyroid and non-thyroid cells. Bovine thyroid fragments were forced through a metallic sieve. The material which filtrates was composed of open cells and cell debris (fraction A); the material remaining on the sieve contained the basal lamina and the interfollicular material as shown by photon and electron microscopic observations (fraction B). Homogenates (HA and HB) were prepared from fractions A and B and centrifuged on a 41% sucrose layer to prepare membrane fractions: MA and MB, which were tested for the presence of adenylate cyclase, TSH-responsive adenylate cyclase and 125I-labelled TSH binding activity. HA and HB contained respectively 70% and 30% of the total thyroid adenylate cyclase activity. MA and MB were similarly enriched in 5'-nucleotidase and adenylate cyclase: 8- to 10-fold as compared to the corresponding homogenates. MA and MB exhibited a marked difference in the response to TSH: TSH either alone or in the presence of Gpp(NH)p stimulated the adenylate cyclase of MA and did not have any effect on MB. Fractionation of MA by isopycnic centrifugation on Percoll gradients yielded a membrane peak exhibiting a TSH-responsive adenylate cyclase activity and a 125I-labelled TSH binding activity displaceable by an excess of unlabelled TSH. A membrane peak at the same density was obtained from MB but its adenylate cyclase did not respond to TSH and there was no specific binding of labelled TSH. Our data indicate that an important fraction of membrane adenylate cyclase of the thyroid does not seem to be coupled with TSH receptor; the major part of this fraction (MB) likely originates from intraglandular non-thyroid epithelial cells. The separation of this membrane fraction from the thyroid cell plasma membrane fraction (MA) allows to increase the response of this latter fraction to TSH.

Activation of adrenal adenyl cyclase by anti-thyroid plasma membrane antibodies.

We have previously shown that IgG isolated from rabbit anti-bovine thyroid plasma membrane (anti-BTPM) antiserum exhibits properties similar to thyroid stimulating antibodies (TSAb) in that it activates thyroid adenyl cyclase. In this study, the organ non-specificity of this reaction was investigated. It was observed that anti-BTPM IgG stimulated not only adenyl cyclase of bovine thyroid but also that of the adrenal. The stimulatory activities on the thyroid and adrenal adenyl cyclase were abolished by absorption of the IgG with bovine adrenal plasma membrane (BAPM). These results indicate that anti-BTPM antibodies, similar to TSAb, exert both thyroidal and extra-thyroidal effects. Thus anti-BTPM antibodies may be directed against antigenic determinants that are common to both thyroid and adrenal plasma membranes. Like the anti-BTPM IgG, anti-BAPM IgG also activated both thyroid and adrenal adenyl cyclase. However, when IgG of the anti-BAPM antiserum was absorbed with thyroid plasma membranes, only the thyroid, but not the adrenal stimulating activity was abolished. It was concluded that the anti-BAPM antiserum contained antibodies directed against membrane antigens specific for the adrenal as well as common antigens shared by the thyroid.

Blocking type antithyrotropin receptor antibody in patients with nongoitrous hypothyroidism: its incidence and characteristics of action.

The incidence, characteristics of action, and pathogenetic importance of blocking type anti-TSH receptor antibody were examined in patients with autoimmune thyroiditis. Serum immunoglobulin G (IgG) from 8 of 20 patients with nongoitrous hypothyroidism contained substantial amounts of TSH binding inhibitor immunoglobulin (TBII) activity. Newborn infants of a patient with the greatest TBII activity had neonatal transient hypothyroidism. In sera of patients with goitrous hypothyroidism and euthryoid chronic thyroiditis, only weakly positive or negative TBII activity was found. IgGs of these patients and those of nongoitrous hypothyroid patients without strongly positive TBII activity did not inhibit TSH stimulation of thyroid adenylate cyclase activity. Seven of 8 IgGs which had strongly positive TBII activity significantly inhibited cAMP generation induced by 9.1 mU/ml TSH, and the eighth IgG inhibited stimulation with 0.5 mU/ml TSH. Although the modes of TSH binding inhibition were variable, markedly close correlation was found between TSH binding- and TSH stimulation-inhibiting activities of these 8 IgGs (r = 0.90; P less than 0.01). These IgGs may exert their inhibitory effects on adenylate cyclase activity by inhibiting TSH binding to its receptor.

Calcium and calmodulin in the regulation of human thyroid adenylate cyclase activity.

TSH (thyrotropin)-stimulated human thyroid adenylate cyclase has a biphasic response to Ca2+, being activated by submicromolar Ca2+ (optimum 22nM), with inhibition at higher concentrations. Calmodulin antagonists caused an inhibition of TSH-stimulated adenylate cyclase in a dose-dependent manner. Inhibition of TSH-and TSIg-(thyroid-stimulating immunoglobulins)-stimulated activity was more marked than that of basal, NaF- or forskolin-stimulated activity. This inhibition was not due to a decreased binding of TSH to its receptor. Addition of pure calmodulin to particulate preparations of human non-toxic goitre which had not been calmodulin-depleted had no effect on adenylate cyclase activity. EGTA was ineffective in removing calmodulin from particulate preparations, but treatment with the tervalent metal ion La3+ resulted in a loss of up to 98% of calmodulin activity from these preparations. Addition of La3+ directly to the adenylate cyclase assay resulted in a partial inhibition of TSH- and NaF-stimulated activity, with 50% inhibition produced by 5.1 microM and 4.0 microM-La3+ respectively. Particulate preparations with La3+ showed a decrease of TSH- and NaF-stimulated adenylate cyclase activity (approx. 40-60%). In La3+-treated preparations there was a decrease in sensitivity of TSH-stimulated adenylate cyclase to Ca2+ over a wide range of Ca2+ concentrations, but most markedly in the region of the optimal stimulatory Ca2+ concentration. In particulate preparations from which endogenous calmodulin had been removed by La3+ treatment, the addition of pure calmodulin caused an increase (73 +/- 22%; mean +/- S.E.M., n = 8) in TSH-stimulated thyroid adenylate cyclase activity. This was seen in 8 out of 13 experiments.

Structure of the thyrotropin receptor and thyroid adenylate cyclase system as determined by target analysis.

Bovine thyroid plasma membranes were irradiated with high-energy electrons. Analysis of the target size of the thyrotropin (TSH) receptor revealed a complex pattern composed of a TSH binding component of 71 000 daltons and a large component (several hundred thousand daltons) that masked some of the binding. Both components were also observed when binding was assayed in the presence of 50 mM NaCl. Membranes preincubated with Mg2+ and 10 microM guanosine 5'-(beta,gamma-imidotriphosphate) [Gpp(NH)p], a persistent activator of adenylate cyclase, also showed the presence of these same components. Although the receptor for TSH has been reported to have some similarities to the receptor for cholera toxin, target analysis of [125I]iodocholera toxin binding was consistent with a single small component about the size of a ganglioside. Measurement of the target size of ground-state, i.e., not preactivated, adenylate cyclase was also carried out. The basal (Mn2+) adenylate cyclase yielded a Mr of 85 000, the smallest unit capable of producing cAMP. The Gpp(NH)p-responsive adenylate cyclase has a Mr of 150 000, which may reflect the contribution of the guanine nucleotide regulatory component to the mass of the active enzyme. A similar size was previously measured for the Gpp(NH)p-preactivated, detergent-solubilized thyroid enzyme [Asbury, R.F., Cook, G.H., & Wolff, J. (1978) J. Biol. Chem. 253, 5286-5292]. Radiation inactivation of the NaF-responsive enzyme indicated two or more components to this activity, the smaller of which (140 000 daltons) was similar in size to the ground-state Gpp(NH)p-responsive enzyme and the larger of which was greater than 10(6) daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of thyroid stimulating immunoglobulins by affinity chromatography using protein A sepharose.

Sera from a reference pool, 29 controls and 18 patients with Graves' disease were fractionated on sepharose CL-4B protein A. After a primary elution of the unbound fraction at normal pH, IgG subclasses 1, 2 and 4 were eluted using a discontinuous pH gradient. The eluate was monitored by UV-absorption at 280 nm and three peaks were observed within narrow pH ranges in all sera tested. From the reference pool (n = 10) peak A was eluted at pH 6.6 +/- 0.1, peak B at 4.2 +/- 0.03 and peak C at 3.8 +/- 0.01. This distribution was partially related to the IgG subclasses with IgG1 recovered in peak C, while IgG2 and IgG4 were distributed in both peak B and C. Thyroid adenylate cyclase stimulating antibodies (TSAb) and thyrotrophin binding inhibiting immunoglobulins (TBII) were measured in the pooled fractions under these peaks. In the reference pool total assay variations (SD) were 10-22 per cent. The 95 percentiles of the 29 control values were used as reference range. In the 18 patients TSAb were positive in 4 in fraction A, 9 in B and 12 in C. TBII were found in one in fraction A, none in B and 13 in C. No correlation between TSAb and TBII values were found before or after fractionation. In most cases TSAb activity was highest in fraction C, however, in 3 patients fraction A was the more active and in these patients TBII were only found in fraction C. It is concluded, that the present results indicate a heterogeneity of the thyroid stimulating immunoglobulins with separation of the TSAb and TBII activities in some patients.

Evidence that organic iodine attenuates the adenosine 3',5'-monophosphate response to thyrotropin stimulation in thyroid tissue by an action at or near the adenylate cyclase catalytic unit.

Studies were conducted to define more clearly the site in the thyroid adenylate cyclase complex at which iodine exerts its inhibitory effect on activation of this enzyme by TSH. Iodine- and TSH-induced desensitization were additive. Dissociation was observed between the rates of recovery from TSH- and iodine-induced desensitization. Cycloheximide (10(-4) M) prevented recovery from the inhibitory effect of iodine on thyroid adenylate cyclase activation. Preincubation of freshly isolated dog thyroid follicles in 10(-4) M iodide decreased the subsequent cAMP response to cholera toxin (0.5 micrograms/ml) stimulation. This effect of iodide was prevented by 3 mM methimazole. Thyroid adenylate cyclase regulatory protein (Ns) activity was assessed by the ability of detergent extracts of thyroid plasma membranes to reconstitute adenylate cyclase responsiveness to isoproterenol in N-deficient S49 cyc- plasma membranes. Thyroid Ns activities were similar in control and iodide-pretreated thyroid cells. The inhibitory effect of iodine on TSH activation of thyroid cAMP generation was additive to that of inhibition via the alpha 2- adrenergic pathway and also additive to inhibition by 2',5'-dideoxyadenosine (an adenosine P-site agonist). Preincubation of freshly dispersed dog thyroid cells in 10(-4) M NaI reduced the cAMP response to stimulation by 100 microM forskolin. These data provide evidence that in iodine-induced TSH desensitization in the thyroid; 1) TSH receptor function is normal, 2) the regulatory protein (Ns) in the adenylate cyclase stimulatory pathway is functionally unaltered, 3) iodine does not exert its effect via the regulatory protein (Ni) in the pathway that inhibits adenylate cyclase activation, 4) iodine does not act via the adenosine P-site inhibitory pathway, 5) the action of iodine is at or near the adenylate cyclase catalytic unit, and 6) new protein synthesis is necessary for recovery from iodine desensitization.

Effect of thyrotropin-induced desensitization of bovine thyroid adenylate cyclase on the nucleotide regulatory protein.

The stimulation of adenylate cyclase by TSH was decreased 50-60% in crude membranes prepared from homogenates of bovine thyroid slices that had previously been incubated for 2 h with the hormone. The diminished response was not associated with any significant change in the binding capacity or affinity for 125I-labeled TSH. The apparent affinities of the desensitized adenylate cyclase for TSH or GTP were not different from those of the enzyme prepared from thyroid slices that had been incubated without TSH. Decreased adenylate cyclase responses to NaF, cholera toxin, or guanyl-5'-yl-imidodiphosphate were also observed in the desensitized membrane, whereas the enzyme responses to prostaglandin E1, GTP, or forskolin were not decreased. However, desensitization caused no decrease in the cholera toxin-catalyzed ADP ribosylation of the 40,000 mol wt polypeptide guanine nucleotide-binding component of the adenylate cyclase. The desensitized membranes showed basal adenylate cyclase activity similar to that of the control membranes using adenyl-5'-yl-imidodiphosphate as substrate in the absence of a nucleotide-regenerating system. These results suggest that the in vitro TSH-induced desensitization of thyroid adenylate cyclase reflects an alteration in the activation processes of the nucleotide regulatory protein.

The prognostic value of parallel measurements of thyrotropin binding inhibiting immunoglobulins (TBII) and thyroid adenylate cyclase stimulating antibodies (TSAb) in Graves' disease after longterm antithyroid treatment.

Thyroid stimulating immunoglobulins were measured in 43 patients with Graves' disease both before and at the end of longterm antithyroid treatment. Parallel determinations were performed of thyrotropin binding inhibiting immunoglobulins (TBII) and thyroid adenylate cyclase stimulating antibodies (TSAb). Before treatment 33 patients were TBII positive and 32 TSAb positive, and at the end of treatment 19 remained TBII positive and 14 TSAb positive. The frequency of relapse was about 70% in the positive patients and about 40% in the patients, who became negative in either test for thyroid stimulating immunoglobulins. By combination of the two assays 23 patients were positive in both before treatment. In these patients 5 relapsed of the 6 who remained positive for both, while none relapsed of the 5 patients, who became negative during treatment. In the remaining 12 patients either TBII or TSAb became negative during treatment and 7 of these relapsed. It is concluded, that the combined measurement of TBII and TSAb in this study seemed superior to the separate determinations of either activity in predicting relapse after medical treatment of Graves' disease, though this evaluation was only possible in part of the patients.