Reorganization of porcine thyroid cells into functional follicles in a chemically defined, serum- and thyrotropin-free medium.

In the serum-free, chemically defined medium NCTC 109, freshly isolated porcine thyroid cells aggregate and form functional follicles in culture even in the absence of thyrotropin. The follicular pattern observed under light and electron microscopy express the main morphological characteristics of in vivo thyroid cells. Follicles are large, replete with dense colloid, and the apical pole of cells is characterized by well-developed microvilli and the presence of aminopeptidase N. The index of iodide transport activity (125I-C/M ratio) decreases vs. days of culture to a resting value of about 1 or 2 at day 2. Addition of thyrotropin (200 microU/ml final concentration) at day 4 is followed by a 10-fold increase in iodide transport activity within 24 h and a 40-fold increase 4 d later. Incorporation and organification of iodide are dose dependent between 0 and 250 microU/ml thyrotropin; highest concentrations (4,000--16,000 muU/ml) are significantly inhibitory. In the absence of thyrotropin each cell synthesizes 8.2 pg thyroglobulin/d. Acute stimulation by thyrotropin at day 4 resulted in a slight decrease in the quantity of thyroglobulin present in the cell layer but in an increase in the total amount of thyroglobulin recovered in both cells and medium, reaching 34.3 pg/cell/d. The protein exported into the medium is thyroglobulin, as shown by SDS PAGE and immunological properties. Here we demonstrate that porcine thyroid cells can be maintained in culture as resting, highly differentiated, follicular-associated cells, sensitive to acute stimulation by thyrotropin.

Endogenous proteolytic activity and constituent polypeptide chains of sheep and pig 19 S thyroglobulin.

Porcine and ovine 19-S thyroglobulins prepared from frozen glands in several buffers using slice extraction or homogenization, ammonium sulfate precipitation and DEAE-cellulose chromatography or Sepharose 6B gel filtration were contaminated with protease activity of pH optima 4.5 and 8.6, as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimum temperatures of autodigestion were 37 degrees C at pH 4.5 and 25 degrees C at pH 8.6. Thyroglobulins prepared from unfrozen glands pH 7.2 in 0.1 M sodium phosphate using slice extraction, ammonium sulfate precipitation and Sepharose 6B gel filtration were devoid of acid proteolytic activity but still underwent autodigestion at pH 8.6. Diisopropylfluorophosphate was a potent inhibitor of the alkaline protease activity of ovine thyroglobulin preparations. In contrast to thyroglobulin obtained from frozen glands the proteins purified from fresh unfrozen glands at pH 7.2 only showed the 19-S and the 12-S species by electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Very few bands migrating faster than 12-S were visible. After full reduction and S-alkylation of porcine and ovine thyroglobulins, no qualitative changes were observed in the gel electrophoresis pattern as compared to the unmodified proteins. Species of apparent mol. wt. corresponding to the native 12 S were the major component, strongly suggesting a mol. wt. of about 330 000 for the elementary peptide chains of pig and sheep thyroglobulins.

Stimulation of sodium and calcium uptake by scorpion toxin in chick embryo heart cells.

Scorpion toxins, the basic miniproteins of scorpion venom, stimulated the passive uptake of Na+ and Ca2+ in chick embryo heart cells. Half-maximum stimulation was obtained for 20-30 nM Na+ and 40-50 nM Ca2+. Scorpion toxin-activated Na+ and Ca2+ uptakes were fully inhibited by tetrodotoxin, a specific inhibitor of the action potential Na+ ionophore in excitable membranes. Half-maximum inhibition was obtained with the same concentration of tetrodotoxin (10 nM) for both Na+ and Ca2+. Scorpion toxin-stimulated Ca2+ uptake was dependent on extracellular Na+ concentration and was not inhibited by Ca2+ channel blocking drugs which are inactive on heart cell action potential. Thus, in heart cells scorpion toxin affects the passive Ca2+ transport, which is coupled to passive Na+ ionphore. Other results suggest that (1) tetrodotoxin and scorpion toxin bind to different sites of the sarcolemma and (2) binding of scorpion toxin to its specific sites may unmask latent tetrodotoxin - sensitive fast channels.

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation.

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into folicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 microU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 microU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.

The effect of triiodothyronine on fatty acid synthetase activity and content in differentiating ob17 preadipocytes.

The effect of triiodothyronine on the activity and amount of the key lipogenic enzyme fatty acid synthetase was studied in differentiating preadipocyte cells (ob17) isolated from ob/ob mouse epididymal fat pad. In the presence of physiological concentrations of insulin, the acquisition of adipose morphology was accompanied by a parallel increase (10--15-fold) in synthetase specific activity and radioimmunoassayable amount relative to soluble cellular proteins. Inclusion of T3 at confluence significantly enhanced synthetase activity and content, with a maximum of 1.5--2-fold above controls at the physiological 1.5 nM concentration, whether insulin is present or not. During adipose conversion, T3 increased the development of enzyme activity and after a longer lag period, the accumulation of the synthetase. Our results suggest that the stimulating effect of T3 upon synthetase activity could involve as a first step the activation of preexisting inactive synthetase molecules and as a second one an increased accumulation of activable synthetase. After longer culture periods, inactive radioimmunoassayable synthetase accumulated.

Effect of carboxypeptidase digestion of the human choriogonadotropin molecule on its thyrotropic activity.

Digestion of hCG by a mixture of carboxypeptidases B and Y results in an enzyme dose- and incubation time-dependent increase in its ability to stimulate the adenylate cyclase (AC) of human thyroid membranes. Treated under the same conditions [3 h at 37 C; enzyme to hCG ratio, 0.04 (wt/wt)], partially and highly purified hCG preparations display an increase of about 300% in thyroid AC-stimulating activity, while TSH displays a 30% decrease. In contrast, carboxypeptidase digestion of hCG under these conditions has no significant effect on its activity in the rat testis AC assay. The carboxypeptidase digestion results in cleavage of carboxyl-terminal amino acid residues 142--145 from the hCG beta-subunit; digestion of the hCG alpha-subunit is much less effective, as the carboxy-terminal amino acid residue 92 is removed from only about 13% of the hCG molecules. In accord with the results of amino acid analysis, a slight, if any, decrease in apparent molecular weight is found by gel filtration and polyacrylamide gel electrophoresis. In addition, carboxypeptidase digestion results in antigenic alterations of the molecule, as shown by a flatter slope of the dose-response curve in a hCG RIA and a 70% decrease in potency in a RIA that uses an antiserum to the hCG beta carboxy-terminal peptide. These data demonstrate that partial digestion of the hCG molecule with carboxypeptidase results in an increase in human thyroid AC-stimulating activity, with retention of the rat testis AC-stimulating activity.

Heterogeneity of the Graves' immunoglobulins directed toward the thyrotropin receptor-adenylate cyclase system.

The TSH-displacing and the thyroid-stimulating activities of Graves' immunoglobulins G (GIgG) have been accounted for by either homogeneous TSH receptor antibodies or heterogeneous antibodies directed toward the TSH receptor-adenylate cyclase system. To clarify this matter, the study of the interactions of GIgG preparations from 14 untreated Graves' patients with human thyroid membranes was undertaken. Dose-response curves of GIgG, diluted in IgG from normal subjects, were carried out in the [125I]TSH radioreceptor assay and the adenylate cyclase assay in the presence or absence of TSH. In the radioreceptor assay, GIgG were constantly negative in 3 cases (21%) and positive, depending of the dose, in 11 cases. In the adenylate cyclase assay, the dose-activity profiles in the absence of TSH were bell-shaped curves in 3 cases and sigmoid curves in 9 cases; in 2 cases (14%), GIgG preparations were devoid of any effect. Binding-isotherms and dose-activity profiles did not appear to share simple relationships. In the presence of TSH, GIgG preparations elicited a decrease in 6 cases, an increase in 3 cases, and no effect in 5 cases (36%) in the adenylate cyclase activity. The data obtained by radioreceptor assay and adenylate cyclase assay in the presence or absence of TSH were found statistically correlated (P less than 0.05 to P less than 0.001) but not linearly related, the points being scattered on specific parts of the diagrams. These observations could not be accounted for by TSH receptor antibodies in GIgG being an entity of constant properties, albeit varying in titer among patients. Rather, GIgG effects fit well the patterns of action of a heterogeneous ligand, as shown by computing a theoretical model for ligand heterogeneity with respect to binding equilibrium constant and intrinsic biological activity. Accordingly, GIgG activity in the TSH receptor-adenylate cyclase system could be attributed to heterogeneous antibodies varying with regard to binding constant and acting as stimulating or blocking antibodies of the adenylate cyclase.

Quantitative in situ hybridization of 3H-labeled complementary deoxyribonucleic acid (cDNA) to the messenger ribonucleic acid of thyroglobulin in human thyroid tissues.

Thyroglobulin (Tgb) mRNA content was studied in human thyroid tissues using liquid hybridization and in situ hybridization. Liquid hybridization revealed no differences in mRNA content, except in the case of colloid adenoma in which a lower amount of Tgb mRNA was found. Conditions for quantitative in situ hybridization of [3H]DNA complementary to the mRNA of Tgb are described. In situ hybridization allowed correlation of the morpho-functional state of the follicles and their content of Tgb mRNA.

Characterization of a hormonogenic domain from human thyroglobulin.

A polypeptide domain of molecular mass near 22 kDa was purified from CNBr-digest of iodine poor human thyroglobulin (hTgb). This fragment represents the N-terminal part of the hTgb molecule and consequently contains the preferential hormonogenic tyrosine 'acceptor' of the protein. This fragment could correspond to the non-iodinated and unreduced form of the thyroxinyl-containing 26 kDa peptide previously purified from reduced and iodinated hTgb. This 22 kDa fragment is capable by itself, i.e. independently of the remaining hTgb molecule, of synthesizing thyroxine with a high efficiency after in vitro iodination. Its study should constitute a valuable way to identify at least one of the hormonogenic tyrosine 'donor' residues of hTgb.

Purification of the human thyroid peroxidase and its identification as the microsomal antigen involved in autoimmune thyroid diseases.

Human thyroid peroxidase (TPO) has been purified from thyroid microsomes by immunoaffinity chromatography using a monoclonal antibody (mAb) to TPO. The eluted material had a specific activity of 381 U/mg and exhibited a peak in the Soret region. The ratio of A411 to A280 ranged from 0.20 to 0.25. Upon SDS-polyacrylamide gel electrophoresis, the purified enzyme gave two contiguous bands in the 100 kDa region. Further, it has been demonstrated that sera with anti-microsomal autoantibodies from patients presenting Graves' or Hashimoto's thyroiditis diseases were able to bind to purified TPO and to inhibit in a dose-dependent manner the mAb binding to purified TPO. This suggests that TPO is the thyroid antigen termed to date the microsomal antigen.

In vitro and in vivo iodination of human thyroglobulin in relation to hormone release.

Reduced and S-alkylated thyroglobulin (Tgb) from different species were shown by SDS-PAGE to contain small peptides (from 45-9 kDa) rich in thyroxine. Several hypotheses were proposed to explain their origin. The polypeptide composition of iodine-poor (Tgb A) and normally iodinated (Tgb B) human Tgb prepared by two different procedures (one minimizing and the other favoring post-mortem proteolysis) was compared in the native state and after in vitro iodination. Results show that one of the hormonogenic sites of human Tgb is part of a domain of the molecule most susceptible to proteolysis, especially when it is very iodinated.

Precursor-product relationship between the 26-kDa and 18-kDa fragments formed by iodination of human thyroglobulin.

At moderate iodination levels (20 iodine at atoms/molecule), human thyroglobulin (hTgb) produces after reduction a thyroxinyl-peptide of 26 kDa which represents the N-terminal part of the protein. At higher iodination levels, the 26-kDa peptide is accompanied by another T4-containing peptide of 18 kDa. A precursor-product relationship between the 26- and 18-kDa fragments was demonstrated by the study of the tryptic fragments of both hormonopeptides. In addition, comparison with the protein sequence deduced from the nucleotide sequence of the 5'-end of hTgb mRNA demonstrated that the N-terminal region of Htgb from which are issued the 26-kDa peptide and its 18-kDa derivative is especially sensitive to proteolysis. This character is possibly related with a facilitated release of thyroid hormones in vivo.

In vitro synthesis of thyroxine in a low molecular weight polypeptide fragment from human thyroglobulin.

A peptide fragment of Mr 16 K was purified from the cyanogen bromide digest of human thyroglobulin either normally iodinated in vivo (0.21 % I) or highly iodinated in vitro (1.40 % I). This peptide segment represents in the native molecule a zone in which tyrosine residues are not or poorly accessible to iodination and consequently do not produce thyroxine. In contrast, after isolation from thyroglobulin and iodination in vitro, the peptide is capable of synthesizing thyroxine with a high efficiency. It is concluded that the peptide described which probably represents a potential hormone forming site in the whole thyroglobulin molecule should constitute a valuable model to study the mechanism of thyroxine formation in vitro.

Thyroglobulin structure and function: recent advances.

Thyroglobulin is a large-size iodoglycoprotein specific to thyroid tissue and is the substrate for the synthesis of thyroid hormones, thyroxine and 3,5,3'-triiodothyronine. Recent studies, which greatly benefited from recombinant DNA methodologies, improved the knowledge of several structural features of this dimeric protein and permitted insights into some structure-function relationships. Analysis-function of the primary structure of the human thyroglobulin monomer revealed several main characteristics: 1) 3 types of internal homologies; 2) extensive homology with the bovine thyroglobulin monomer and known partial sequences in the thyroglobulins of other mammalian species; 3) significant homologies with 2 other non-thyroid proteins (acetylcholinesterase and the invariant chain of the Ia class II histocompatibility antigen); 4) a terminal localization of the hormonogenic sites at both ends of the monomer. Current studies aim at determining conformational characteristics, understanding the molecular mechanisms of thyroid hormone formation and unraveling those interactions which in the thyroid cell and the thyroid follicle will permit this large pro-hormone to synthesize and release a few small thyroid hormone molecules. A more precise knowledge of this molecule in higher vertebrates and during evolution would impart valuable information concerning thyroid pathology, since thyroglobulin has been implicated in some genetic and in autoimmune thyroid diseases.

Idiotypic analysis of five xenogeneic antisera to anti-human thyroglobulin monoclonal antibodies.

Five xenogeneic anti-idiotypic antisera (anti-id) were produced against individual BALB/c-derived monoclonal antibodies (mAb) which bound to different peptidic determinants on the human thyroglobulin molecule (Tg). Idiotypic analysis performed using sensitive radioimmunoassays revealed that: (1) the anti-id highly precipitated their homologous ligands; (2) two anti-id displayed minor cross-reactivities with one or two heterologous mAb; (3) each unlabelled homologous mAb was able to inhibit the idiotype binding of the corresponding anti-id; (4) no significant inhibition of homologous idiotype binding was observed with large excess of heterologous mAb; (5) efficient inhibition of mAb binding to Tg was observed only when homologous anti-id served as inhibitor. The data support the conclusion that xenogeneic anti-id may detect on their corresponding ligands individual idiotypic specificities that can be located at the mAb-combining site. Such reagents may constitute appropriate probes for further studies on anti-Tg autoimmunity.

Follicle formation and iodide metabolism in cultures of human thyroid cells.

Primary cultures were initiated using thyroid tissue obtained at operation from patients with Graves's disease. The in-vitro conditions which permitted the formation of functional follicular structures in both primary cultures and derived sub-cultures were examined. In both situations, culture without the addition of calf serum to the medium resulted in the formation of follicles in response to thyrotrophin. In primary cultures the response to stimulation by exogenous thyrotrophin was variable. However, cells derived from long-term primary monolayers responded to thyrotrophin stimulation in a more predictable manner. In sub-cultures, the ability of cells to concentrate and organify iodide was augmented in a dose-dependent fashion in response to thyrotrophin (0 to 0.2 mu./ml); maximal values of 20 to 80 times those of control cultures being obtained. While follicular structure was maintained at higher hormone concentrations iodide-trapping capacity declined. Similar effects were produced by both low and high purity thyrotrophin and by dibutyryl cyclic AMP. Thyroid cells from two patients with a genetic defect of iodide organification exhibited the same lesion in vitro.

Thyrotropin-specific binding to human peripheral blood monocytes and polymorphonuclear leukocytes.

[125I]Labeled thyrotropin binding to leukocytes has been studied using lymphocyte-, monocyte- and polymorphonuclear leukocytes-enriched preparations obtained by centrifugation of Ficoll-Angiocontrix gradients and Sephadex G-10 adherence. From the relation between thyrotropin binding and phagocytosis as shown by latex beads ingestion, it is concluded that the hormone binds essentially to monocytes and polymorphonuclear leukocytes. Equilibrium association constants of the high-affinity, low-capacity sites (1 nM-1) are similar to those found in isolated thyroid cells or in thyroid plasma membranes. The role of thyrotropin in the regulation of phagocytosis by leukocytes is discussed.

Thyrotropin-induced plasma membrane protein modifications in porcine thyroid cells.

Highly purified plasma membranes were obtained from isolated porcine thyroid cells maintained in conditions of culture in the presence of thyrotropin (stimulated cells) or in their absence (non-stimulated cells). Analyses of both types of membranes by high-resolution sodium dodecylsulfate-polyacrylamide slab gel electrophoresis showed reproducible quantitative differences in protein bands of apparent molecular weight 38,000, 36,000 and inconstantly 96,000. Phosphorylation of membranes by [gamma-32P]ATP was 2-3 times higher in membranes from thyrotropin-stimulated than in membranes from non-stimulated cells. About 20 32P-labeled bands were detected by slab gel electrophoresis in denaturing conditions, among which the catalytic subunit of Na+, K+ ATPase was characterized. In addition, plasma membranes from thyrotropin-stimulated cells contained a firmly bound [14C]glucosamine-containing glycoprotein probably related to an aggregation-promoting factor. 125I-labeled thyroglobulin and components of unknown nature were associated with plasma membranes from thyrotropin-stimulated cells. Whether they participate in the structure and function(s) of the plasma membrane or represent contaminants of the preparation is not clear at the present time.

Chemical composition of porcine thyroid cell plasma membranes.

Plasma membranes were isolated from thyroid cells obtained by trypsinization of porcine glands and maintained in culture conditions in the presence or absence of thyrotropin or dibutyryl cyclic AMP. The protein, phospholipid, cholesterol and sialic acid content of the 3 types of cell plasma membranes were very similar. High cholesterol and sialic acid content characterized these membranes. The amino acid and carbohydrate composition was similar to that shown for other eukaryotic plasma membranes. Sodium dodecylsulfate-polyacrylamide gel electrophoresis disclosed the presence of more than 20 protein bands, of which six corresponded to glycoproteins.

Phosphorylation of purified thyroid plasma membranes incubated with [32-P]ATP.

Plasma membranes have been prepared from porcine thyroid glands using sucrose gradients. The fractions having a density in sucrose of 1.18 g/ml mainly contained plasma membranes and were moderately contaminated with other subcellular components as shown by marker enzyme data. Purified plasma membranes incubated in the presence of [32-P]gamma ATP incorporated 32-P. Kinetics of incorporation of 32-P into endogenous substrates studied in various buffers and with increasing ATP concentration suggest a phosphodephosphorylating system related to cAMP-dependent protein kinase and phosphoprotein phosphatase activities. The two enzymatic activities associated with plasma membranes have been demonstrated using exogenous substrates. cAMP increases and fluoride ions decrease the extent of membrane phosphorylation. The specific activity of protein kinase was 10-12 times higher than in the initial homogenate and was only slightly enhanced in the presence of 0.5% Nonidet as compared to microsomal fraction. cAMP binding to membrane proteins was 3 times higher than to the other particulate fractions. TSH present in the incubating medium or added after 5 min of 32-P labelling induced a rapid stimulation of endogenous phosphorylation followed by a rapid decrease. Phosphorylated membrane substrates were analyzed: high voltage paper electrophoresis after partial hydrolysis indicated that [32-P]phosphate is incorporated into serine and threonine residues as o-phosphate derivatives. SDS-polyacrylamide gel electrophoresis showed several 32--labelled fractions. When enhanced by cAMP, no specific phosphorylation of protein components was observed.