Studies of the congenitally goitrous sheep. Iodoproteins of the goitre

1. Congenitally goitrous thyroid tissue was obtained from South Australian Merino sheep. Ultrastructural studies of the secretory cells in this tissue showed active cells of normal appearance, containing apical protein droplets. 2. (125)I-labelling in vivo of goitre tissue was used to investigate the iodoproteins, in which the major proportion of (125)I appeared in the cell protein fraction soluble in 0.9% sodium chloride (average 62% in goitres from untreated sheep). 3. Ammonium sulphate fractionation showed two clear peaks of iodoprotein precipitation, one at 35-40% saturation and the other at 50-55% saturation. Both iodoprotein fractions contained iodotyrosines and iodothyronines, which were identified chromatographically after enzymic hydrolysis of the protein. 4. Polyacrylamide-gel electrophoresis at pH9.4, at either 7.5 or 5.0% acrylamide concentration, was used to characterize the iodoproteins. Two major fractions were observed, the fastest-migrating fraction coincident with serum albumin, and a slower-migrating, less-well-defined zone. This fraction migrated in 7.5% acrylamide gel, which excluded normal thyroglobulin. 5. Density-gradient (10-40% sucrose) centrifugation was used to determine the approximate sedimentation coefficients of the iodoproteins, which showed major components at s(20,w) 8-9S and s(20,w)<5S. 6. Immunoprecipitation with rabbit anti-(sheep thyroglobulin) failed to sediment (125)I-labelled proteins from goitre extracts. 7. Ouchterlony-type double diffusion in agar plates demonstrated immunoprecipitation lines between rabbit anti-(sheep thyroglobulin) and both the concentrated goitre extract and its Sephadex G-200-excluded fraction, which were confluent with that obtained on reaction with purified normal thyroglobulin. 8. It was concluded that both major iodoprotein fractions were capable of supplying thyroid hormones to the animal, and that the fraction of s(20,w)<5S was iodinated serum albumin. As (125)I-labelled thyroglobulin was not detected in goitre tissue from untreated or thyroxine-treated animals, it was possible that the genetic defect causing goitre resulted in an abnormal thyroglobulin, incapable of being iodinated but immunologically reactive.     

Thyroglobulin interactions with thyroid plasma membranes. The existence of specific receptors and their potential role.

Thyroglobulin binds to isolated thyroid plasma membrane preparations. Binding is pH- and temperature-dependent with 10-fold better binding at pH 5.0 and 37 degrees C than at 0 degrees C and pH 6.0 through pH 7.5. Binding is, however, maximal in 90 min at all pH values and temperatures examined. Although salts can inhibit or enhance thyroglobulin binding depending on the temperature or pH, conditions approaching those of the physiological state are not inhibitory; physiological conditions do inhibit thyrotropin binding to the same membrane preparations. 125I-Labeled thyroglobulin binding is poorly reversed by unlabeled thyroglobulin at all pH values and temperatures studied; excess unlabeled thyroglobulin can, however, readily prevent binding. At pH values greater than 6.0 and at 0 degrees C, the iodine content of thyroglobulin can affect binding, and the 27 S thyroid iodoprotein is relatively ineffective in preventing the binding of the 19 S species. At pH 5.0 and 37 degrees C, there is no difference in binding of highly and less iodinated thyroglobulin, and the 27 S thyroglobulin iodoprotein is effective in preventing 19 S thyroglobulin binding. The complex nature of these results is interpreted in the light of additional data which show (i) that the thyroid membrane recognizes asialothyroglobulin and (ii) that at pH 5.0 and 37 degrees C a membrane-associated neuraminidase is activated which removes sialic acid from thyroglobulin. Vibrio cholerae neuraminidase can substitute for the endogenous neuraminidase. The receptor on thyroid membranes for asialothyroglobulin is similar to the asialoglycoprotein receptor on liver membranes (Morell, A.G., Gregoriadis, G., Scheinberg, I.H., Hickman, J., and Ashwell, G. (1971) J. Biol. Chem. 246, 1461-1467) in that sialic acid on the receptor is critical for receptor expression. It is distinct from the liver asialoglycoprotein receptor in its binding specificity and in its sensitivity to different bacterial and mammalian neuraminidase preparations. Relationships between thyroglobulin and thyrotropin receptors on thyroid membranes are explored, and the functional role of the thyroglobulin receptor is discussed.     

Immunochemical and immunohistochemical studies on the 27 S iodoprotein of dog thyroid with reference to thyroglobulin-like reaction of the parafollicular cells.

Our earlier finding that the thyroglobulin-like material responsible for the immunoreaction of parafollicular cells obtained in peak I fraction of Bio-Gel A-5m was followed up in the present study by an investigation of the immunochemical and immunohistochemical reactions of 27 S iodoprotein which was the most prominent material in the peak I fraction. The antibody was raised against completely purified 27 S iodoprotein which was obtained as follows: Thyroglobulin was extracted from dog thyroids and chromatographed initially on Bio-Gel A-5m and then on Bio-Gel A-50m. The area of 27 S migrated as a single bank on polyacrylamide gel slab electrophoresis. This was cut and eluted. Anti-27 S antiserum showed the same immunochemical patterns to 27 S and 19 S as anti-19 S antiserum with three different immunochemical methods: double diffusion test, one dimensional and two dimensional immunoelectrophoresis. The immunoperoxidase reactions of the anti-27 S antiserum and anti-19 S antiserum were restricted to follicular cells and luminal colloids. No reaction of the parafollicular cells was obtained by these antisera. Thus, 27 S iodoprotein shared common immunochemical and immunohistochemical properties with 19 S thyroglobulin. It was concluded that 27 S iodoprotein was not responsible for the thyroglobulin-like reaction of the parafollicular cells.     

Process of iodination of thyroglobulin and its maturation. I. Properties and distribution of thyroglobulin labeled with radioiodine in pig thyroid slices.

Pig thyroid slices were incubated with Na131I and the 17--19S 131I-labeled thyroglobulin isolated was subjected to dissociation with 0.3 mM sodium dodecyl sulphate SDS) on sucrose density gradient centrifugation and to iodoamino acid analysis. During the incubation, initially dissociable thyroglobulin was gradually altered to 0.3 mM SDS-resistant species with increasing incorporation of iodine. Microsome-bound, poorly iodinated thyroglobulin and preformed thyroglobulin were chemically iodinated and then subjected to analysis of dissociability and iodoamino acid contents with newly incorporated iodine. The results indicated that the behavior of the former thyroglobulin resembled that of 131I-thyroglobulin obtained from the slices. Then, thyroid slices were incubated for 3 min with Na131I and 3H-leucine with or without 10-min chase incubation. The sucrose density gradient centrifugation patterns of 131I and 3H-radioactivity of cytoplasmic extracts indicated that 131I-thyroglobulin is contained in particulates, especially in vesicles with low density(d=1.12) and that some of them are released into the soluble fraction within 10 min. The vesicles contained peroxidase and NADH-cytochrome c reductase, and are probably exocytotic vesicles in the apical area of cytoplasm of follicular cells. No positive evidence was obtained that plasma membranes participate in the iodination of thyroglobulin under the present experimental conditions. These results suggest that, in the incubation of thyroid slices, iodine atoms are preferentially incorporated into newly synthesized, less iodinated thyroglobulin, rather than preformed thyroglobulin, and that the iodination occurs, at least to a certain degree, in apical vesicles before the thyroglobulin is secreted into the colloid lumen.     

Subunit structure of 27 S thyroid iodoprotein.

The dissociation of thyroid 27 S iodoprotein by sodium dodecyl sulfate (SDS) and by succinic anhydride was investigated by means of ultracentrifugation and polyacrylamide gel electrophoresis. The iodoprotein obtained from either a human or hog was dissociated into three kinds of subunits (S-19, S-17 and S-12) by SDS treatment. At increased concentrations of SDS, the S-12 subunit was predominant among the dissociation products. The succinylation of 27 S iodoprotein showed essentially the same dissociation pattern as in the case of SDS treatment. This dissociation products of the protein preparations of different animals were qualitatively the same as those of thyroglobulin of the respective animals, confirming the hypothesis that 27 S iodoprotein was composed of two molecules of thyroglobulin. However, the extent of dissociation of 27 S iodoprotein measured by S-12 formation showed higher resistancy of the protein to the dissociating agents than that of thyroglobulin. The contents of sialic acid and hexose as well as iodoamino acids of 27 S iodoprotein were found to be the same as, or not far from, those of thyroglobulin; The dissociability and chemical composition of 27 S iodoprotein was discussed with reference to the subunit structure of the protein.     

Isolation and characterization of hagfish thyroid iodoprotein by its non-thyroglobulin nature, very high iodine and carbohydrate contents and low hormone/iodine ratio

We have characterized the thyroid iodoprotein of a hagfish, Eptatretus burgeri, one of the lowest marine vertebrates. The iodoprotein was not very homogeneous in its apparent molecular mass which decreased with the increase in hormone/iodotyrosine ratio. Four subfractions with an apparent molecular mass of about 400 kDa were purified from one major fraction by size-exclusion and Mono Q ion-exchange HPLC. The subfractions appeared to have the same peptide backbone, since they showed a single band with the same mobility as a 160-kDa protein in SDS/PAGE and the same amino acid composition. However they differed from each other in having increasing iodine contents (1.9% to 5.9% by mass of total amino acids) associated with the increase in hormonal iodine proportion (8.4% to 16.7% of total iodine) and carbohydrate content (35.6% to 53.5% by mass). These values are strikingly different from those of thyroglobulin with an iodine content of less than 1%, hormonal iodine of 20-40% and carbohydrate content of less than 10%. The amino acid composition of the hagfish iodoprotein, especially the cysteine content of less than 1%, was also entirely different from that of thyroglobulin. These results suggest that most, if not all, tyrosine residues of the hagfish thyroid glycoprotein with a less rigid structure are susceptible to an iodinating system, but hormone residues are formed by a much less efficient mechanism than those in thyroglobulin, when poorly iodinated.     

Immunohistochemical study of a large molecular fragment of thyroglobulin in parafollicular cells

Thyroglobulin-like immunoreactivity of the parafollicular cells was studied by an immunoperoxidase bridge technique using antisera against dog thyroglobulin fragments. 1. The dog parafollicular cells were specifically stained by anti-peak I (27S and larger components fraction) antiserum absorbed with peak II (19S fraction). By this method, they were easily distinguishable from the non-reactive follicular cells and colloid droplets. More sensitive staining of the parafollicular cells was possible with anti-peak I' (larger components fraction) antiserum. The staining reactions indicated that the antigenic material responsible for immunoreactivity of the parafollicular cells was due to larger molecular components of thyroglobulin corresponding to 32S, 37S or greater than 37S, and was not due to either the 19S thyroglobulin or to the 27S iodoprotein. 2. A conspicuous decrease of the immunoreactive material in the parafollicular cells occurred in the dog after both chronically induced hypercalcemia and antithyroid drug treatment. This coincided with movement of secretory granules containing calcitonin as shown by staining with silver impregnation, HCl-basic dye, and lead-hematoxylin. 3. The antisera against larger molecular components of dog thyroglobulin showed a high degree of cross-reactivity to the parafollicular cells of most of the mammalian species investigated; rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human.     

Process of iodination of thyroglobulin and its maturation. II. Properties and distribution of thyroglobulin labeled in vitro or in vivo with radioiodine, 3H-tyrosine, or 3H-galactose in rat thyroid glands.

With the aim of obtaining information on the process of iodination of thyroglobulin, the properties and subcellular distribution of thyroglobulin labeled with radioiodine, 3H-tyrosine, or 3H-galactose were studied. The following results were obtained for 17-19S thyroglobulin isolated from rat thyroid lobes labeled in vitro. (a) The effect of sodium dodecyl sulfate (SDS) concentration (0.1-2.0 mM) on the dissociability of the proteins into 12S subunits showed that 3H-labeled, 131I-labeled, and preformed thyroglobulin behaved very differently; their dissociability decreased in that order. In addition, 0.3 mM SDS is most suitable for discriminating among these species. (b) The amount of 0.3 mM SDS-resistant 131I-thyroglobulin increased with the time of incubation of the lobes or with the amount of iodine atoms incorporated by chemical iodination. (c) Digestion of 3H-tyrosine-labeled thyroglobulin showed that 3H-monoiodotyrosine and 3H-diiodotyrosine were present after incubation of the lobes for 180 min. (d) The dissociability of 3H-galactose-labeled 17-19S thyroglobulin was higher than that of 131I-labeled protein, but its elution pattern on DEAE-cellulose chromatography resembled that of the latter. (e) 131I-Thyroglobulin was scarcely found in the incubation medium, although a considerable amount of 19S thyroglobulin was released into the medium during the incubation. As for the lobes, a significant amount of 131I-radioactivity as well as 3H-radioactivity was found in cytoplasmic particulates, especially in fractions containing apical vesicles and rough microsomes. On the other hand, the following results were obtained for 17-19S thyroglobulin isolated from rats injected with 125I. (a) Dissociability of the protein by 0.3 mM SDS and analysis of 125I-iodoamino acids of pronase digest showed that the iodination process was essentially similar to the case of in vitro incorporation, but was faster. (b) The effect of cyclohiximide treatment showed that the relative reduction of 0.3 mM SDS dissociable species was probably due to a shortage of newly synthesized proteins. All the results obtained in the present experiments are compatible with the view that iodine atoms are incorporated selectively into newly synthesized, less iodinated thyroglobulin, and that the iodination occurs intracellularly, at least to a certain degree, after carbohydrate attachment, probably in the apical vesicles. The possibility that iodination also occurs to some extent in the endoplasmic reticulum and in the colloid lumen of thyroglobulin-stimulated thyroids is discussed.     

Change of the protein composition of the thyroid colloid during treatment with propylthiouracil and thyroxine A microgel electrophoretic study of single rat thyroid follicles

Propylthiouracil and thyroxine were given daily to rats for 4 weeks. Samples of colloid were collected in vivo from the superficial thyroid follicles during this period and their protein composition was analysed by gel electrophoresis.It was observed that the aggregates of thyroglobulin, i.e., the 27-S thyroid iodoprotein and the heavier fractions, were reduced to 50% after 1 week and were almost absent after 2 weeks.A faster migrating thyroglobulin fraction was observed in the samples of colloid and in the homogenate of the whole gland after 48 h of treatment. During the following period of treatment there was an increase in the relative amount of the faster migrating thyroglobulin fraction compared to 19-S thyroglobulin in the colloid, the former comprising approx. 75% of the globulins after 4 weeks, It can be concluded that propylthiouracil inhibits the formation of the 27-S iodoprotein and that a structurally altered and iodine-poor thyroglobulin fraction is accumulated in the follicle lumen.     

Change of the protein composition of the thyroid colloid during treatment with propylthiouracil and thyroxine: a microgel electrophoretic study of single rat thyroid follicles

Prophylthiouracil and thyroxine were given daily to rats for 4 weeks. Samples of colloid were collected in vivo from the superficial thyroid follicles during this period and their protein composition was analysed by gel electrophoresis. It was observed that the aggregates of thyroglobulin, i.e., the 27-S thyroid iodoprotein and the heavier fractions, were reduced to 50% after 1 week and were almost absent after 2 weeks. A faster migrating thyroglobulin fraction was observed in the samples of colloid and in the homogenate of the whole gland after 48 h of treatment. During the following period of treatment there was an increase in the relative amount of the faster migrating thyroglobulin fraction compared to 19-S thyroglobulin in the colloid, the former comprising approx. 75% of the globulins after 4 weeks, It can be concluded that propylthiouracil inhibits the formation of the 27-S iodoprotein and that a structurally altered and iodine-poor thyroglobulin fraction is accumulated in the follicle lumen.     

The effect of iodide administration on hog thyroid gland and the composition of thyroglobulin and 27-S iodoprotein.

The effect of excess iodide on hog thyroid gland has been examined with regard to the change in the chemical composition of thyroglobulin and in the accumulation of 27-S iodoprotein by the in vivo treatment of hogs with iodide for various lengths of time. The iodine content of thyroglobulin was either unchanged by short term administration of excess iodide, or somewhat lowered. However, the iodine content as well as the total amount of thyroglobulin increased in the glands enlarged by prolonged treatment with iodide. The iodine highest reached 1.17% of the protein on an average. On the other hand, 27-S iodoprotein decreased and finally disappeared after the chronic treatment. Monoiodotyrosine and diiodotyrosine increased in parallel with the increase in the iodine content (0.15 to 1.17%) caused by the iodide treatment, while thyroxine increased but reached a plateau at the level of three residues per mole of thyroglobulin, and no change was observed even in the proteins with the higher iodine content than 0.75%. Proteolytic activity measured by amino acid release from the thyroid protein was depressed by the chronic treatment. On the other hand, the amount of iodocompound released by the autoproteolysis, which may reflect hormone secretion, increased, possibly because of the marked increase in the iodine content of thyroglobulin.     

Iodinated phospholipids and the iodination of proteins of dog thyroid gland in vitro

Slices of dog thyroid gland were incubated with liposomes consisting of (125)I-labelled phosphatidylcholine (the iodine was covalently linked to unsaturated fatty acyl chains). The (125)I label of (125)I-labelled liposomes was incorporated into thyroid protein and/or thyroglobulin at a higher rate than was the (131)I label of either Na(131)I or (131)I(2). The iodine was shown to be protein-bound by the co-migration of the labelled iodine with protein under conditions where free iodine, iodide and lipid-bound iodine were removed from protein. The uptake of iodine from the iodinated phospholipid was probably due to phospholipid exchange between the iodinated liposomes and the thyroid cell membrane, since (a) (14)C-labelled phospholipid was metabolized to (14)CO(2) and (b) many lipids in the tissue slice became (14)C-labelled. A very strong inhibition of iodide ;uptake' from Na(131)I, caused by thiosulphate, produced only a minor inhibition of the incorporation of (125)I from (125)I-labelled liposomes into thyroid protein and/or thyroglobulin. This implies that free iodide may not necessarily be formed from the iodinated phospholipids before their entrance or utilization in the cell. Synthetic polytyrosine polypeptide suspensions showed some iodination by (131)I-labelled liposomes. In tissues with low tyrosine contents, such as liver and kidney, only a trace uptake was observed. Salivary gland showed some uptake. Endoplasmic reticulum of thyroid gland showed a higher iodine uptake than that of the corresponding plasma membranes. These experiments, together with the demonstration of the diet-dependent presence of iodinated phospholipids in dog thyroid, leads us to suggest that iodination of the membrane phospholipids of thyroid cells may be directly or indirectly involved at some stage in the synthesis of thyroglobulin, or exists as a scavenger mechanism, to re-utilize and/or recover released iodine from unstable compounds inside the thyroid cell.     

Iodoamino acid composition of rat thyroglobulin of varying total iodine concentration and fractions isolated by isopycnic ultracentrifugation]

Iodoaminoacid content (iodothyronines, T3 and T4, and iodotyrosines, MIT and DIT) has been determined in enzymatic hydrolysates of thyroglobulin Tg 19S of different iodine content (0.3-0.9%) isolated from equilibrium labeled rats. Preparative equilibrium centrifugation in RbCl density gradients of pure thyroglobulin was used to obtain protein fractions of largely different iodine content (0.2-1.2% I). Thin layer chromatography of total hydrolysates demonstrated that the distribution of iodoaminoacids depends on the total iodine content of each fraction. It is concluded, in agreement with previous results, that the native structure of Tg is an important factor in the regulation of hormone biosynthesis and that even at low iodination levels of Tg. T3 and T4 are synthesized.     

A comparative review of the structure and biosynthesis of thyroglobulin

Thyroglobulin, the major iodoglycoprotein of the thyroid (M_r 669 kDa) has a sedimentation coefficient of 19 S and an isoelectric point (pI) of 4.4–4.7. The protein has been isolated and purified from saline extracts of the gland of several animal species, by methods such as ammonium sulfate fractionation, DEAE-cellulose chromatography and Sepharose 4B/6B gel-filtration. DEAE-cellulose chromatography of thyroglobulin from many species, by linear gradient, yielded a complex elution pattern, while camel thyroglobulin showed only a major and minor peak. As an iodoprotein, the protein has 0.1–2.0% iodine. The amino acid and iodoamino acid composition of thyroglobulins, in general, is similar. However, a high thyroxine content (15 mol/mol protein) has been noted for buffalo species. Asparagine or aspartic acid has been reported as the major N-terminal amino acid for thyroglobulins of several animal species whereas glutamic acid is the sole N-terminal amino acid for buffalo thyroglobulin. As a glycoprotein, thyroglobulin contains 8–10% total carbohydrate with galactose, mannose, fucose, N-acetyl glucosamine and sialic acid residues. The carbohydrate in the protein is distributed as two distinct units, A and B. In addition, human thyroglobulin has carbohydrate unit C. The occurrence of sulfate and phosphate as Gal-3-SO₄ and Man-6-PO₄, respectively, has been reported in few species. The quaternary structure of native thyroglobulin is comprised of two equal sized subunits of 330 kDa. However, the protein appears to contain 4–8 non-identical units in few species. The synthesis of thyroid hormones occurs in the matrix of the protein and is regulated by pituitary thyrotropin. The role of tyrosine residues 5 and 130 in thyroxine synthesis has been well documented.     

Thyroglobulin biosynthesis in a larval (ammocoete) and adult freshwater lamprey (Lampetra planeri Bl.).

1. The biosynthesis of 18-19S thyroglobulin has been studied in a larval and adult freshwater lamprey (Lampetra planeri Bl.). 2. In vivo and in vitro experiments have been performed by injecting into the coelomic cavity or by incubating branchial region labeled constituents of Tg of higher vertebrates (125I, [3H]leucine and various [3H]carbohydrates). 3. Larvae (ammocoetes) and adults incorporate all labels into thyroglobulin (18-19S Tg), containing a small proportion of labeled T3 and T4, as identified by paper chromatography, and very minute amounts of stable iodine. 4. In adults, the biosynthesis of 18-19S Tg proceeds much more rapidly and the labels are incorporated in higher percentage than in larvae. 5. The demonstration of the biosynthesis of the specific thyroid protein, 18-19S Tg, in larvae indicates that the biochemical mechanism of hormonogenesis is present in larval endostyle before the morphological differentiation of thyroid cells and follicles occurring during metamorphosis. 6. Some 18-19S Tg is apparently stored in the endostyle.     

Use of Methotrexate to Treat Isolated Graves Ophthalmopathy Developing Years After Thyroidectomy and Iodine 131 Treatment of Papillary Thyroid Cancer

ObjectiveTo describe a case of Graves ophthalmopathy developing years after subtotal thyroidectomy and radioactive iodine treatment of papillary thyroid cancer.MethodsWe present a case report including clinical and laboratory data. Current relevant literature is reviewed and summarized with regard to Graves ophthalmopathy.ResultsIn 2001, a 51-year-old woman presented with an asymptomatic thyroid nodule. Fine-needle aspiration biopsy results showed Hürthle cells, and the patient had a subtotal thyroidectomy in 2002. Stage 2 follicular variant of papillary thyroid carcinoma was diagnosed. She received radioactive iodine (I 131) therapy (94.8 mCi and 147.2 mCi) in 2003. Thyrotropin was suppressed with levothyroxine. The patient remained asymptomatic and had undetectable thyroglobulin antibodies. In 2007, her eyes became irritated (ie, erythematous, pruritic, watery). Thyroperoxidase and thyroglobulin antibodies were undetectable, but thyrotropin receptor antibody was elevated to 44% (reference range, < 16%). On physical examination, moderate periorbital edema and conjunctival injection were present; orbital magnetic resonance imaging was normal. Computed tomography of her orbits showed symmetric bilateral exophthalmos and prominence of orbital fat. Other ophthalmologic etiologies were ruled out by 2 independent ophthalmologists. She had minimal improvement with oral and intravenous steroids. Subsequent treatment with methotrexate resulted in marked symptomatic improvement and lowered the thyrotropin receptor antibody level to 24%.ConclusionsIsolated Graves ophthalmopathy in a patient after treatment of thyroid cancer and radioactive iodine ablation has not been previously reported. Methotrexate therapy may be a useful therapeutic approach in this setting. (Endocr Pract. 2008;14:422-425)     

Evidence for a secretion of thyroxine by isolated hog thyroid cells.

Isolated thyroid cells prepared from hog thyroid glands by tryptic dispersion were incubated with 131I- for 1--6 h. Free [131I]thyroxine was identified in the incubation medium by three chromatographic methods. Neither [131I]iodotyrosines nor [131I]triiodothyronine were detected. The [131I]thyroxine released in the medium by 100 mul of cells (packed cell volume) after a 6-h incubation period amounted to 1.16% (S.E. = +/- 0.39) of the total radioactivity. The medium [131I]thyroxine represented 15--25% of the total [131I]thyroxine synthesized during the 6 h of incubation. Thyrotropin, 1--60 munits/ml, increased the medium [131I]thyroxine content 2-4 fold. Dibutyryl cyclic AMP mimicked the effect of thyrotropin. The amount of medium [131]thyroxine was strictly related to the amount of incubated cells but was independent of the volume of the incubation medium. When prelabeled cells were incubated in the presence of methimazole the increase in medium [131I]thyroxine was quantitatively related to a decrease in the intracellular [131I]thyroxine. Addition of dinitrotyrosine, an inhibitor of the deiodinase activity, induced the release of iodotyrosines in the incubation medium. That the incubation supernatant of isolated thyroid cells did contain free thyroxine but not iodotyrosines suggests that the normal mechanisms of proteolysis of thyroglobulin and deiodination of iodotyrosines inside the cells are preserved. From these data, it was concluded that the thyroxine release by isolated cells represents a real secretion.     

Different sedimentation properties of agonist- and antagonist-labelled platelet alpha2 adrenergic receptors

Alpha₂ adrenergic receptors were solubilized from human platelet particulate preparations with digitonin. The solubilized alpha₂ receptors retained the essential binding specificity characteristics of the membrane-bound receptors. The alpha₂ receptors could be labelled in platelet membranes with either agonist ([³H]epinephrine) or antagonist ([³H]yohimbine) radioligands. When these membranes were solubilized with digitonin and centrifuged on sucrose density gradients, the sedimentation coefficient of the agonist-labelled receptor (14.6S) was greater than that of the antagonist-labelled receptor (12.9S). This observation may provide insight into the mechanism of adenylate cyclase inhibition by alpha₂ adrenergic receptors.     

The subcellular distribution of 32P-labelled phospholipids, 32P-labelled ribonucleic acid and 125I-labelled iodoprotein in pig thyroid slices. Effect in vitro of thyrotrophic hormone and dibutyryl-3′,5′-(cyclic)-adenosine monophosphate

1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.     

The inhibition of PB125I formation in calf thyroid caused by 14-iodo-15-hydroxy-eicosatrienoic acid is due to decreased H2O2 availability

Previous work from our laboratory has shown that 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-HO-A) is a potent inhibitor of iodine organification in calf thyroid slices. The present studies were performed in order to clarify the mechanism of this action. Incubation of thyroid slices with 10(-4)M I-HO-A caused a 47 and 53% decrease in PB125I formation after 30 and 60 min incubation, respectively. In a series of experiments an inverse relationship between the degree of inhibition caused by I-HO-A and total iodine content and basal iodoprotein formation was observed. Chromatographic analysis of the labeled compounds showed a significant decrease in 125I incorporation into MIT, DIT, T3 and total iodolipid. The site of the inhibitory effect of I-HO-A was then sought. TPO was measured by three different methods. When TPO was solubilized from I-HO-A treated slices, no change in enzymatic activity was observed. Moreover, the same lack of action was found when solubilized TPO was incubated with I-HO-A. The production and release of H2O2 into the incubation medium was measured by chemiluminiscence technique. In control slices the values increased during the first 10 min and reached a plateau. Pretreatment of the slices with 10(-4)M KI caused a 51% inhibition, while the same concentration of I-HO-A produced a 59% inhibition. The possibility that I-HO-A might exert its action through a putative protein inhibitor was also explored. Incubation of slices with 10(-5)M I-HO-A caused a 46% decrease in PB125I formation and addition of actinomycin D or puromycin failed to alter this effect.(ABSTRACT TRUNCATED AT 250 WORDS)