Pendred syndrome maps to chromosome 7q21-34 and is caused by an intrinsic defect in thyroid iodine organification.

Exactly 100 years ago, in 1896, Pendred first described the association of congenital deafness with thyroid goitre (MM#274600). The incidence of Pendred syndrome is estimated at 7.5-10/100,000, and may be responsible for as much as 10% of hereditary deafness. The cause of the congenital deafness in Pendred syndrome is obscure, although a Mondini type malformation of the cochlea exists in some patients. The reason for the association between the thyroid and cochlear defects is similarly obscure, leading some investigators to suggest that the two recessive defects may be occurring together by chance in highly consanguineous families. An in vivo defect in thyroid iodine organification in Pendred syndrome patients has been reported. However, the molecular basis of this defect is unknown and the presence of an intrinsic thyroidal defect has not been conclusively demonstrated. We have adopted a genetic linkage study as a first step towards identifying the gene. The availability of an inbred Pendred syndrome kindred allowed us to utilize an efficient DNA pooling strategy to perform a genome-wide linkage search for the disease locus. In this way, we have mapped the disease locus to an approximately 9-cM interval between GATA23F5 and D7S687 on chromosome 7. In addition, we demonstrate an intrinsic thyroid iodine organification defect in a patient's thyroid cells as the cause of the thyroid dysfunction.

Control of HLA-DR antigen expression by gamma-interferon: separate signal transduction mechanisms in malignant and nonmalignant human thyroid cells.

Three intracellular signal transduction pathways have been found to be utilized by gamma-interferon (IFN-gamma) in the induction of HLA-DR in several cell types, mainly monocytes/macrophages and B-cells: the protein kinase A (PKA); Ca(2+)-calmodulin; and protein kinase C (PKC) pathways. In this study, we investigated the role of these pathways in IFN-gamma-induced HLA-DR expression in normal and neoplastic human thyroid cells. The PKA pathway seemed to inhibit both neoplastic and normal IFN-gamma-induced HLA-DR expression; addition of thyroid-stimulating hormone to normal thyroid cells, as well as 8-bromo cyclic AMP and forskolin to normal and neoplastic cells, reduced the amount of IFN-gamma-induced HLA-DR. Moreover, H-8, a PKA inhibitor, enhanced such IFN-gamma-induced HLA-DR expression. The calcium-calmodulin pathway does not seem to play a role in IFN-gamma-induced HLA-DR expression in normal and neoplastic thyrocytes, since the Ca-ionophore A23187, EGTA, and the calmodulin antagonist, W-7, neither induced HLA-DR nor showed any effect on HLA-DR expression induced by IFN-gamma. Alone, phorbol 12-myristate 13-acetate, a PKC activator, did not induce HLA-DR on thyroid cells. However, its addition to neoplastic cells together with IFN-gamma caused a synergistic elevation of the expressed HLA-DR, whereas it significantly inhibited IFN-gamma-induced HLA-DR in normal thyrocytes. TPA had to be added before or together with IFN-gamma for optimal function. If added more than 6 h after IFN-gamma, TPA was not effective. An inactive TPA analogue did not affect HLA-DR induction, while an active analogue mimicked TPA. Staurosporine, a PKC inhibitor, reduced the TPA enhancing effect in neoplastic thyrocytes and cancelled TPA inhibition in normal cells. Moreover, when added to IFN-gamma without TPA in normal thyroid cells, staurosporine increased 3- to 4-fold the amount of HLA-DR. Thus, in normal thyroid cells the PKC pathway is activated by IFN-gamma and inhibits HLA-DR expression. In neoplastic thyrocytes, although IFN-gamma does not induce HLA-DR via PKC, this pathway augments HLA-DR expression.

Role of the carboxy-terminal residues of the alpha-subunit in the expression and bioactivity of human thyroid-stimulating hormone.

The glycoprotein hormones TSH, CG, LH, and FSH are heterodimers consisting of a hormone-specific beta-subunit and a common alpha-subunit. The aim of the present study was to investigate the role of the carboxy terminus of the common alpha-subunit (amino acids Tyr89-His90-Lys91-Ser92), which has been shown to be important for human (h) CG and hFSH, for the activity of hTSH. Successive truncations of the alpha-carboxy terminus by site-directed mutagenesis revealed a stepwise reduction of bioactivity occurring at residues alpha Ser92 and alpha His90 to 64% and 13%, respectively. This contrasts with previous findings for hCG and hFSH, where loss of bioactivity occurred in a single step with the deletion of alpha Lys91 but alpha Ser92 was not important. The decreased bioactivities of the hTSH alpha-truncation mutants were reflected by concomitant reductions of cAMP production, thyroid hormone synthesis and cell growth and were accompanied by a loss of receptor binding. Substitution of residues alpha Lys91 or alpha His90 with either a hydrophobic or a bulkier residues resulted in a reduction of receptor binding and signal transduction, indicating that the alpha-carboxy terminus of hTSH may interact with the TSH receptor in a tight contact area. Conversely, substitution of alpha His90 with smaller residues enhanced bioactivity. In addition, the integrity of the alpha-carboxy terminus was essential for hTSH expression. Thus, we showed common and different roles of the alpha-carboxy-terminal residues for the glycoprotein hormones. The unique role of alpha Ser92 in hTSH activity explains the evolutionary constraint to preserve the alpha-carboxy-terminal Ser92 in all glycoprotein hormones.

The role of the asparagine-linked oligosaccharides of the alpha-subunit in human thyrotropin bioactivity.

TSH and the gonadotropins (FSH, LH, and hCG) are a family of heterodimeric proteins that share a common alpha-subunit and differ in their hormone-specific beta-subunit. The asparagine-linked (N-linked) oligosaccharides on these hormones are important in signal transduction. The N-linked oligosaccharides on the alpha-subunit have no effect on hCG and hFSH receptor binding, but are critical for their biological activity. Here, we analyzed the role of alpha-subunit N-linked oligosaccharides in human TSH (hTSH) bioactivity by site-directed mutagenesis and gene transfer. This was achieved by mutating the asparagine (Asn) residue in the N-linked glycosylation consensus sequence (Asn-X-Thr/Ser) to aspartic acid. The wild-type hTSH and its variants were expressed in Chinese hamster ovary cells. Wild-type alpha-subunit and its mutants (alpha 1, alpha 2, and alpha(1 + 2)) were efficiently combined with TSH beta-subunit and secreted as dimers. The bioactivity of TSH glycosylation variants was determined by measuring their abilities to stimulate cAMP formation and T3 secretion using a serum-free culture system of human thyroid follicles. Using this system, wild-type hTSH was significantly effective in the stimulation of cAMP formation and T3 secretion. Deletion of the oligosaccharide units from either site 1(alpha 1) or site 2(alpha 2) of the alpha-subunit increased the biological activity of the dimer by about 30%. However, deletion of carbohydrate units from both sites of hTSH alpha-subunit (alpha(1 + 2) resulted in a significant reduction in cAMP formation (by approximately 70%) and T3 secretion (by approximately 40%) compared to that with wild-type hTSH. These findings emphasize the importance of the alpha-subunit N-linked oligosaccharide chains on hTSH bioactivity.

Mutual antagonistic interactions between the thyrotropin (adenosine 3',5'-monophosphate) and protein kinase C/epidermal growth factor (tyrosine kinase) pathways in cell proliferation and differentiation of cultured human thyroid follicles.

Our aim has been to delineate the role of the major signal transduction pathways believed implicated in the control of thyroid function and growth: the cAMP-, epidermal growth factor (EGF) (tyrosine kinase)-, and protein kinase C (PKC)-mediated mechanisms. The experimental model used was our system of thyroid follicles of human origin cultured in suspension under serum-free conditions in which the follicular three-dimensional structure is retained. The phorbol ester 12-O tetradecanoylphorbol 13-acetate (TPA) time and dose dependently (10(-11)-10(-7) M) inhibited TSH-stimulated thyroid functions (cAMP formation, iodide uptake and organification, and T3 secretion). TPA also inhibited such forskolin- and 8-BrcAMP-stimulated effects, suggesting that the attenuation of the cAMP-dependent pathway occurs at steps both pre- and post-cAMP formation. The effects of TPA were mimicked by another PKC activator, phorbol 12,13-dibutyrate, but not by a phorbol ester that fails to activate PKC, 4 alpha-phorbol 12,13-didecanoate, and were reversed by staurosporine, a PKC inhibitor. The TPA actions seem, therefore, to be PKC-mediated. EGF exhibited a time- and dose-dependent (0.02-8 nM) restraining influence on the above TSH-stimulated differentiated functions, except for cAMP, which was enhanced. EGF also blunted such forskolin- and 8-BrcAMP-induced response parameters, suggesting inhibition at a post-cAMP locus. Regarding cell proliferation, during the initial stages of culture (day 2), TPA dose dependently (10(-11)-10(-7) M) attenuated cell proliferation, but subsequently (day 7 of culture) the same doses of TPA stimulated cell multiplication. The TPA-mitogenic and antimitogenic effects could not be mimicked by 4 alpha-phorbol-12,13-didecanoate and were reversed by staurosporine, thus indicating a PKC-mediated pathway for such TPA actions. EGF, on the other hand, only enhanced cell proliferation at a late stage (coincident with the TPA-mitogenic effect). TSH (0.5 U/liter) inhibited both the mitogenic and antimitogenic actions of TPA as well as the cell-proliferative influence of EGF. In conclusion, the data demonstrate mutual antagonistic interactions between the signal transduction pathways: the PKC and EGF (tyrosine kinase) pathways seem to inhibit TSH (cAMP)-mediated human thyroid cell differentiation, whereas TSH attenuates PKC-mediated thyroid cell mitogenesis and antimitogenesis as well as EGF-mediated cell proliferation.

The protein kinase A pathway inhibits c-jun and c-fos protooncogene expression induced by the protein kinase C and tyrosine kinase pathways in cultured human thyroid follicles.

We have previously demonstrated antagonistic interactions between the major signal transduction pathways in human thyroid follicles: TSH acting via protein kinase A (PKA) attenuated phorbol ester [acting via protein kinase C (PKC)] as well as epidermal growth factor (EGF)-protein tyrosine kinase (PTK)-mediated cell proliferation, whereas the PKC and PTK pathways inhibited PKA-mediated cell differentiation. In view of the key role played by the protooncogenes c-jun and c-fos in the cascade of events leading to cell proliferation and differentiation, we examined whether the antagonism we observed between the pathways could be related to changes in the expression of these genes. The experimental model used was the same in vitro system as that used in the above study on cell growth and differentiation: thyroid follicles of human origin cultured in suspension under serum-free conditions. Both EGF (1-50 ng/mL) and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 10(-11)-10(-7) mol/L) dose and time dependently stimulated c-jun and c-fos messenger ribonucleic acid (mRNA) expression. The c-jun and c-fos mRNA stimulation elicited by TPA was reduced by the PKC inhibitors, chelerythrine and staurosporine, and could not be mimicked by 4alpha-phorbol 12,13-didecanoate (a phorbol ester that fails to activate PKC), whereas the stimulation induced by EGF was diminished by the PTK inhibitor, genistein. This indicates a PKC- and PTK-mediated pathway triggered by TPA and EGF, respectively. TSH induced an increase in c-jun and c-fos mRNA, which, though significant, was small compared to that elicited by TPA or EGF. Addition of TSH (0.1-0.5 mU/mL), however, to either TPA or EGF dose dependently inhibited the c-jun and c-fos mRNA elicited by these agents. The repressive action of TSH on the effects of TPA and EGF mRNA were mimicked by forskolin and 8-bromo-cAMP, suggesting that the TSH inhibitory action is PKA mediated. The TSH inhibitory action seems to require de novo protein synthesis, as it was abrogated in the presence of cycloheximide. In conclusion, the present study provides novel data on c-jun and c-fos gene expression and their modulation by the major signal transduction pathways operating in human thyrocytes. Moreover, using the same serum-free system of human thyroid follicles cultured with the same agents and at the same doses as in our previous study on cell growth and differentiation, we found the TSH/PKA pathway to inhibit PKC- and EGF/tyrosine kinase-induced c-jun and c-fos mRNA, i.e. antagonistic effects parallel to those previously observed measuring cell proliferation. The findings suggest an association between human thyroid cell proliferation and c-jun and c-fos gene expression.

Thyrotropin, acting at least partially via adenosine 3',5'-monophosphate, exerts both mitogenic and antimitogenic effects in cultured human thyroid cells.

The aim of this study was to examine the role of TSH and cAMP in human thyroid cell proliferation. For this purpose, human thyrocytes were cultured in the absence or presence of TSH, cAMP analogs, or forskolin, and the following parameters were measured during 7 consecutive days: [3H]thymidine incorporation into trichloroacetic acid-precipitable material, cell count (to assess cell proliferation), and cAMP accumulation. Cells proliferated during the first days in culture, reaching a maximum on day 5, followed by a decline in cell growth. TSH dose-dependently enhanced thyrocyte proliferation during the first few days of culture, but attenuated cell growth during the later stages of culture. The TSH mitogenic action was only apparent at a low cell density (10(4) cells/well in 96-well microtiter plates). TSH elicited a dose-dependent elevation of cAMP during all phases of cell culture. Furthermore, the cAMP analogs, 8-bromo-cAMP and dibutyryl cAMP, mimicked the thyrocyte proliferative and antiproliferative actions of TSH. Forskolin also mimicked the TSH mitogenic and antimitogenic effects while concomitantly elevating cAMP levels during both instances. The data, therefore, seem entirely consistent with the premise that the TSH stimulatory and inhibitory actions on human thyroid cell growth are mediated, at least partially, by cAMP. The dual actions of TSH as a mitogenic and antimitogenic factor may, thus, provide an experimental in vitro basis for the well established in vivo goitrogenic effect of TSH observed initially, followed by desensitization on prolonged exposure to the hormone.

Similar and divergent patterns in the regulation of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of MMP-1 gene expression in benign and malignant human thyroid cells.

An imbalance between the activity of matrix metalloproteinases (MMPs) (proteolytic enzymes that degrade protein components of the extracellular matrix) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), may be one of the mechanisms responsible for tumor cell invasion. We have investigated the regulation of MMP-1 and TIMP-1 gene expression in benign and malignant (follicular, anaplastic, and papillary) human thyroid cells. As expected of cells with invasive potential, detectable MMP-1 messenger RNA (mRNA) levels were observed in malignant cells under basal conditions, in contrast to undetectable levels in benign cells. Exposure of these cells, for 1 h, to the active phorbol ester, phorbol 12-myristate 13-acetate (TPA, 100 nmol/L), acting via protein kinase C (PKC), elicited an increase in MMP-1 mRNA, with a peak stimulation after a 3- to 4-h culture period. Epidermal growth factor (EGF, 25 ng/mL), however, acting via protein tyrosine kinase (PTK), stimulated such gene expression in malignant cells but failed to do so in benign cells. TIMP-1 mRNA was not significantly altered by the TPA-PKC, EGF-PTK, or TSH-protein kinase A (PKA) pathways in malignant cells. In benign cells, however, TPA induced a small, though significant, increase in TIMP-1. The MMP-1 stimulation by EGF and lack of TPA-induced rise in TIMP-1 in malignant cells, in sharp contrast to the effects obtained in benign thyrocytes, seems to indicate that the MMP: TIMP balance favors a more extensive extracellular matrix protein breakdown by malignant thyrocytes, as expected of cells exhibiting invasive capacity. TSH (10-500 microU/mL) failed to significantly influence basal MMP-1 or TIMP-1 mRNA levels, but it caused a dose-dependent inhibition in TPA- and EGF-induced MMP-1 mRNA in malignant cells, and TPA-stimulated MMP-1 and TIMP-1 in benign cells. The repressive action of TSH on MMP-1 mRNA was mimicked by forskolin and 8-bromo-cAMP and was abrogated by the PKA inhibitor, H-89, suggesting that the TSH inhibitory action is PKA-mediated. In conclusion, the present study provides novel data on MMP-1 and TIMP-1 gene expression and their modulation by the major signal transduction pathways operating in human thyroid cells. Similar and divergent patterns have emerged in the regulation of such gene expression in benign and malignant human thyrocytes, in many instances in accord with the concept of MMP playing the role of stimulating, and TIMP inhibiting, cell invasion. Although MMP-1 may be just one of the many factors responsible for tumor cell invasion, the present findings demonstrating the possibility, at least in vitro, of repressing MMP gene expression may have important clinical ramifications.

Triiodothyronine and follicle-stimulating hormone, alone and additively together, stimulate production of the tissue inhibitor of metalloproteinases-1 in cultured human luteinized granulosa cells.

Thyroid disorders have been frequently associated with menstrual disturbances and impaired fertility. To characterize the nature of thyroid hormone action in the ovary, the direct effects of T3-gonadotropin interactions were investigated in vitro using a culture system of human luteinized granulosa cells in serum-free medium. Although FSH alone was devoid of any significant effect on cell proliferation, it inhibited T3-stimulated cell growth. The electrophoretic profiles of the radiolabeled proteins induced by the different hormonal treatments revealed similarity in overall protein patterns but differences in intensity of labeling. Human CG, alone or combined with T3, had no major influence on the total intensity of labeling compared with control, whereas T3 or FSH alone reduced total labeling intensity but a 30,000 Da protein band was increased. FSH combined with T3 augmented the total intensity of labeling, including the 30,000-Da protein band. Western blot analysis revealed the presence of the tissue inhibitor of metalloproteinases-1 (TIMP-1), mol wt 30,000, known to play a key role in ovarian function. TIMP-1 was dose dependently stimulated by T3 and FSH, and an additive effect was obtained when both hormones were combined. This is the first report of TIMP-1 modulation by FSH in ovarian cells and of an effect by thyroid hormone on TIMP-1 levels. The study shows TIMP-1 induction in human ovarian cells not only by FSH, i.e. via a probable protein kinase A mechanism, but also demonstrates an additional mode of TIMP-1 hormonal induction: via thyroid hormone stimulation, acting by modulation of gene transcription. The present study provides novel data on TIMP-1 hormonal modulation and of direct T3 in vitro ovarian effects that may account for the in vivo indications of a thyroid-ovarian connection.

Human chorionic gonadotropin stimulates thyroid hormone secretion, iodide uptake, organification, and adenosine 3',5'-monophosphate formation in cultured human thyrocytes.

Despite extensive studies, the issue of whether hCG possesses intrinsic thyrotropic activity remains unresolved. This is mainly because in the experimental systems used so far, the parameters measured did not include the thyroid-specific functions of iodine organification and the hormonal end-point response, T3 secretion, and cells of nonhuman origin were employed, constituting a major drawback in view of the wide variation in sensitivity of thyroid responsiveness to hCG in different species. We investigated the thyrotropic activity of hCG, using for this purpose a novel homologous assay system consisting of human thyroid follicles cultured suspended in collagen gel in serum-free medium. Under these conditions, the cells are organized as follicular three-dimensional structures with normal polarity, enabling enhanced responsiveness to hormonal stimulation. The parameters measured were the thyroid-specific functions of iodide uptake, organification, and T3 secretion, as well as formation of the second messenger, cAMP. Purified hCG (biological potency, 21,700 IU/mg; with no detectable TSH by immunoradiometric TSH assay) did indeed exhibit thyroid stimulatory activity. At doses ranging from 10-400 mg/L, hCG induced a dose-dependent increase in the parameters measured. The rise from basal to maximal levels achieved after hCG stimulation was 1.3 to 3.6 pmol/well for cAMP formation, 34 to 21,408 cpm/well for iodide uptake, 261 to 20,167 cpm/well for iodide organification, and 40 to 927 fmol/well for T3 secretion. Maximal levels elicited by hCG (200 mg/L) relative to maximal values achieved with bovine TSH were 49%, 56%, and 42% for iodide uptake, organification, and T3 secretion, respectively, and only 5% for cAMP. Iodide uptake proved to be the most sensitive indicator of the thyrotropic activity of hCG, with increases occurring at a concentration of 10 mg/L. Acting as a partial agonist, hCG was also capable of dose-dependently inhibiting TSH-stimulated cAMP formation. The free alpha- and beta- subunits of hCG, at doses as high as 200 mg/L, had no thyroid-stimulating effect. The present data thus clearly demonstrate that hCG is a human thyroid stimulator. Moreover, hCG managed to elicit substantial biological cell responses in human thyrocytes while evoking minimal amounts of cAMP, illustrating the concept of cAMP superfluity and highlighting the potential pitfalls of using cAMP as a reliable measure of hormonal bioactivity.

Effects of gamma-interferon on DR antigen expression, growth, 3,5,3'-triiodothyronine secretion, iodide uptake, and cyclic adenosine 3',5'-monophosphate accumulation in cultured human thyroid cells.

We have examined, using the same system of human thyroid cells in culture, the effects of the cytokine human gamma-interferon (gamma IFN) on the expression of DR antigen, cell pro-liferation, cAMP accumulation, and the differentiated functions, iodide uptake and T3 secretion. gamma IFN elicited a dose- and time-dependent increase in DR expression, with a maximum effect on day 5 of culture. The cytokine, at the same concentrations and experimental conditions as those found to be effective in inducing DR antigen expression, caused on day 5 of culture a dose-dependent inhibition of [3H]thymidine incorporation, DNA content, cell count, as well as TSH-stimulated (but not basal) iodide uptake and T3 secretion. The gamma IFN suppressive influence on the differentiated functions was not merely due to a reduction in cell number, but was also apparent when results were expressed per micrograms DNA. Since the cytokine did not inhibit TSH- or forskolin-stimulated cAMP accumulation and showed a suppressive influence toward 8-bromo-cAMP- and forskolin-stimulated T3 secretion, its inhibitory effect seems to be exerted at a site located distal to cAMP formation. Although gamma IFN alone was devoid of any effect on cAMP accumulation, it enhanced forskolin-stimulated (as well as TSH-activated) cAMP in the presence of 3-isobutyl-1-methylxanthine, an inhibitor of cAMP degradation. Thus, it would seem that gamma IFN also exerts an influence on cAMP formation (rather than degradation) at a step subsequent to TSH binding to its receptor. The effects we observed seem specific to gamma IFN, since alpha IFN, although capable of inhibiting human thyrocyte multiplication, lacked any influence on DR antigen expression, cAMP accumulation, or T3 secretion by human thyroid cells. In what way, if any, is gamma IFN-induced DR antigen expression on human thyrocytes, an event believed to be critical in the pathophysiology of autoimmune thyroid disease, related to decreased thyroid function and growth is presently unknown.

Sulfate transport is not impaired in pendred syndrome thyrocytes.

Pendred syndrome is the most common form of syndromic deafness, characterized by dyshormonogenic goiter associated with sensory-neural deafness. The gene responsible for the disease (PDS) has been cloned, but its function is as yet unknown and the connection between thyroid goiter and sensory-neural deafness remains an enigma. PDS codes for a novel protein, pendrin, which is closely related to a number of sufate transporters. Mechanisms by which abnormal sulfate transport could deleteriously affect iodide organification have been proposed. We tested sulfate transport in thyrocytes obtained from Pendred syndrome patients and found that it was not defective. This suggests that pendrin in fact may not be a sulfate transporter, and emphasizes the importance of functional studies on this novel protein.

Toxic multinodular goiter: a variant of autoimmune hyperthyroidism.

The aim of this study was to examine whether at least a subgroup of patients with toxic multinodular goiter may have autoimmune thyroid disease. Thyroid-stimulating immunoglobulin (TSI) activity, measured by a sensitive bioassay employing cultured human thyroid cells, was determined in patients with toxic multinodular goiter and other thyroid disorders. All patients with active Graves' disease (n = 47) had detectable serum TSI activity, whereas TSI was undetectable in patients with thyroid disease not believed to be of autoimmune origin: toxic adenoma (n = 13), cold nodule (n = 5), and nontoxic goiter (n = 19), with a single exception in the latter group. Toxic multinodular goiter (n = 26) was diagnosed based on clinical and laboratory evidence of hyperthyroidism associated with a multinodular goiter on palpation and scintiscan. The toxic multinodular goiter group was then subclassified according to scintiscan pattern (type A, diffuse but uneven distribution of technetium uptake; type B, multiple discrete nodules of varying size and function). All but 1 of the 11 TSI-positive toxic multinodular goiter patients had a type A scintiscan pattern. The patients with the type A scintiscan pattern were younger and more often had elevated antithyroid antibody titers, ophthalmopathy, and concurrent development of goiter and hyperthyroidism (rather than long-standing goiter preceding hyperthyroidism) compared to the type B patients. Thus, a subgroup of patients with clinically defined toxic multinodular goiter (type A) probably have autoimmune hyperthyroidism (a variant of Graves' disease), while in another subgroup (type B) hyperthyroidism is not related to an autoimmune etiology (a variant of toxic adenoma).

Effect of acute cimetidine administration on indices of parathyroid hormone action in healthy subjects and patients with primary and secondary hyperparathyroidism.

To evaluate the acute effect of histamine H2-receptor blockade with cimetidine on PTH concentrations and its endogenous biological activity, we studied the effect of iv administration of cimetidine (50-mg bolus followed by 500 mg/60 min infusion) in normal subjects and patients with primary and secondary hyperparathyroidism. No significant changes in serum concentrations of calcium, phosphate, or immunoreactive C-terminal PTH were found in any group. However, the control group had decreased calciuria, increased phosphaturia, and decreased tubular reabsorption of phosphate, while the primary hyperparathyroid group had increased phosphaturia, increased nephrogenous cAMP excretion, and decreased tubular reabsorption of phosphate. In both of these groups, the finding suggest an increase in PTH-like biological activity. These findings suggest that cimetidine is not useful in the diagnosis or medical management of hyperparathyroidism. In fact, the changes in renal calcium and phosphorous excretion indicative of PTH-like biological activity along with a decrease in creatinine clearance in the primary hyperparathyroid group suggest a relative contraindication to the use of this drug in these patients.

Monoamine oxidase activity and triiodothyronine biosynthesis in human cultured thyroid cells.

1. The proposal that monoamine oxidase (MAO) is a source of peroxide in thyroid hormone biosynthesis has been examined by use of isolated cultured human thyroid cells which retain the ability to secrete triiodothyronine (T3) in response to thyroid stimulating hormone (TSH). 2. The results demonstrated the presence of MAO A and B in human thyroid cells which oxidized 5-hydroxytryptamine and 2-phenylethylamine, respectively, and were selectively inhibited by the MAO inhibitors clorgyline and (-)-deprenyl. 3. Addition of propylthiouracil to the culture system induced a 61% reduction in TSH-stimulated T3 secretion, indicating that the bulk of such secretion apparently derives from de novo iodothyronine synthesis. 4. The MAO A and B substrate, tyramine, was ineffective in stimulating T3 secretion. 5. The selective MAO inhibitors, clorgyline and (-)-deprenyl, alone and in combination, and in the presence and absence of tyramine, failed to inhibit basal as well as TSH-stimulated T3 secretion in cultured human thyrocytes. 6. It is therefore apparent that even though thyroid MAO A and B enzyme reactions result in the generation of H2O2, this H2O2 does not seem to play a significant role in T3 biosynthesis.

Dexamethasone and 8-bromo-cyclic AMP depress the incorporation of [3H]thymidine into mouse condylar cartilage by different pathways.

We examined whether cyclic AMP (cAMP) affects the incorporation of [3H]thymidine into cartilage cells and, if so, whether this action could be related to the inhibitory effect of glucocorticoid hormones on the growth of ossifying cartilage. Incorporation of [3H]thymidine into trichloroacetic acid-precipitable material by mouse cartilage was measured concomitantly with the concentration of cAMP. Dexamethasone (1 mumol/l) significantly (P less than 0.05) depressed the incorporation of [3H]thymidine. The cAMP analogue 8-bromo-cAMP (0.01-1 mmol/l) also depressed the incorporation of the radionucleotide in a dose-dependent fashion. When various concentrations of 8-bromo-cAMP were added with dexamethasone (1 mumol/l), no apparent changes took place compared with the effect of dexamethasone alone. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.2-1 mmol/l) elicited an inhibitory effect on [3H]thymidine incorporation and a stimulatory influence on cartilage cAMP concentrations. Dexamethasone, at doses (0.01-1 mumol/l) causing significant inhibition of [3H]thymidine incorporation, failed to increase cartilage levels of cAMP. It seems, therefore, that the depressive effect of dexamethasone on [3H]thymidine incorporation in condylar cartilage is not mediated through an increase of cAMP in the tissue.

Iodide uptake and organification, tri-iodothyronine secretion, cyclic AMP accumulation and cell proliferation in an optimized system of human thyroid follicles cultured in collagen gel suspended in serum-free medium.

An experimental system was established for measuring cell function and proliferation of human thyroid follicles cultured in collagen gel suspended in serum-free medium. Optimal culture conditions were defined and the system was characterized. The human thyrocytes were functional as indicated by their ability to respond to a TSH stimulus (as low as 1-10 microU/ml), in a time- and dose-dependent fashion, with at least a 15-fold increase in iodide uptake and organification, tri-iodothyronine (T3) secretion (demonstrated to derive from de-novo T3 biosynthesis) and cyclic AMP accumulation. Moreover, the same system allowed the measurement of cell proliferation (as indicated by thymidine incorporation and DNA content) following epidermal growth factor (EGF) and phorbol ester challenge under conditions of cell density and medium identical to those for the differentiated functions. The functional responses and cell proliferation were markedly higher compared with those of the same cells in the presence of serum or maintained in monolayer culture. Normal cell polarity, which critically determines functional capacity of thyroid follicles was maintained (as demonstrated by electron microscopy) by the use of collagen gel and serum-free medium. The use of thyroid cells of human origin assumes great importance in view of the wide species differences reported. Cryopreservation of cells rather than the necessity of using freshly derived cells confers greater convenience. The present model system provides a powerful tool for studying human thyroid physiology and pathophysiology.

Sensitization and desensitization of human thyroid cells in culture: effects of thyrotrophin and thyroid-stimulating immunoglobulin.

Using an in-vitro system of cultured human thyroid cells and cyclic AMP (cAMP) accumulation as an index of cell stimulation, we compared TSH and thyroid-stimulating immunoglobulin (TSI) with regard to thyrocyte sensitization and desensitization. The smallest dose of TSH (0.05 mU/ml) capable of stimulating thyroid cells was the same as the minimum dose required to induce desensitization upon subsequent rechallenge with the hormone. In contrast, about 30-fold higher doses of TSI were needed to cause cell refractoriness compared with doses capable of eliciting stimulation. Moreover, significant stimulation of the thyroid with TSI was apparent much later than with TSH. A longer time-lapse was also necessary for TSI to induce desensitization. Likewise, thyrocytes recovered more slowly from TSI compared with TSH desensitization. Although at high doses TSI induced homologous desensitization, at lower doses the antibody, unlike TSH, potentiated the cAMP response to subsequent exposure to the antibody. The stimulatory doses of TSI were in the range usually encountered in active Graves' disease, which may explain why prolonged TSI in vivo sustains a hyperthyroid condition. In addition, we found that under conditions in which TSH leads to desensitization of the cAMP response, the thyroid cells maintained their responsiveness in terms of triiodothyronine secretory activity. Pre-exposure of human thyrocytes to TSI induced heterologous desensitization towards the TSH-stimulated cAMP response. Moreover, addition of the antibody to maximally desensitizing doses of TSH decreased cell sensitivity to the hormone even further.(ABSTRACT TRUNCATED AT 250 WORDS)

Triiodothyronine (T3) modulates hCG-regulated progesterone secretion, cAMP accumulation and DNA content in cultured human luteinized granulosa cells.

Ample clinical evidence indicates that women with thyroid disorders frequently exhibit menstrual disturbances and impaired fertility. In order to characterize the nature of thyroid hormone action in the ovary, the direct effects of triiodothyronine (T3) were investigated in vitro using a culture system of human luteinized granulosa cells. The presence of T3 receptors was also searched in such cells. The cell cultures were maintained in serum-free Ham's F-10 medium in the absence or presence of hCG, with or without graded doses of T3 (10(-11)-10(-7) M), and cell proliferation (assessed by DNA content) as well as cell function (cAMP accumulation and progesterone secretion) determined. T3 alone stimulated cell proliferation. hCG, on the other hand, was anti-mitogenic and T3 combined with hCG inhibited cell growth even further, reaching levels below those reached by either control or hCG alone. Exposure of cells to T3 markedly enhanced hCG-induced cAMP accumulation. Addition of 1-methyl-3-isobutylxanthine (MIX) abolished the cAMP-stimulatory effect elicited by T3, suggesting that the thyroid hormone may act, as MIX, by inhibiting phosphodiesterase. T3 was devoid of any influence on basal progesterone secretion, but inhibited hCG-induced secretion of the steroid. The effects of T3 are not accounted for by changes in cell number since the influence of thyroid hormone on cAMP and steroid secretion were expressed per microgram DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolactin inhibits hCG-stimulated steroidogenesis and cAMP accumulation, possibly by increasing phosphodiesterase activity, in rat granulosa cell cultures.

The effects of prolactin (PRL), alone and together with human chorionic gonadotropin (hCG), on steroidogenesis and cAMP accumulation in the preovulatory ovary were studied. Cultured granulosa cells obtained from large preovulatory follicles of pregnant mare serum gonadotropin (PMSG)-treated immature rats were used. The results indicated that PRL inhibited, in a dose-dependent manner, hCG-induced cAMP accumulation and 17 beta-estradiol (E2) secretion. When added to 0.4 IU/ml hCG (designated as 100% activity), 1, 10 and 100 ng/ml PRL decreased cAMP accumulation to 86, 64 and 59%, respectively, following 1 h incubation and to 87, 81 and 66% E2 secretion, respectively, following 48 h incubation. PRL alone failed to cause any significant change in cAMP or E2 concentrations. The inhibition of PRL was apparently not at the hCG receptor level, since a similar inhibitory effect was observed in prostaglandin E1 (PGE1)-induced cAMP accumulation. Nor was the inhibitory pathway of adenylate cyclase involved, since pertussis toxin--an inactivator of the Gi regulatory protein--failed to abolish the suppressive effect of PRL on hCG-induced cAMP accumulation. The phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methyl-xanthine, abolished the inhibitory effect of PRL on hCG- and PGE1-induced cAMP accumulation and on hCG-induced E2 secretion, indicating that PRL might be inhibiting cAMP accumulation and steroidogenesis in preovulatory granulosa cells by enhancement of PDE activity.