A method for selective infusion in the thyroid artery of the rat and mouse.

A technique for in vivo infusion in the superior thyroid artery in rats and mice was developed and evaluated. The influx catheter is inserted in retrograde direction into the superior carotid artery. The infusate mixed with blood is directed exclusively to the thyroid lobe via the superior thyroid artery. The thyroid isthmus is divided and the other lobe serves as a control. Thyroid ultrastructure was unaltered after infusion for at least 4 h and the follicle cells displayed a normal morphological response to TSH. Electron microscopical autoradiography (125I, [3H]leucine) was performed using 20-80 times less label as compared with iv administration. Infusion of forskolin, a stimulator of adenylate cyclase, increased the intrathyroidal cyclic AMP levels about 10-fold. Infusion of the ionophore monensin yielded typical dilations of Golgi cisternae as well as reduced secretion of newly synthesized protein into the follicle lumen. The arterial infusion technique developed is useful when in vitro methods or systemic administration of substances are unsuitable. The technique permits selective administration of small amounts of experimental substances to the thyroid in high concentrations.

Forskolin-induced elevation of cyclic AMP does not cause exocytosis and endocytosis in rat thyroid follicle cells in vivo.

Using a newly developed infusion technique, the in vivo effects of forskolin and dibuturyl cyclic AMP (dbcAMP) on exocytosis and endocytosis in thyroid follicle cells were studied in thyroxine-treated rats and mice. Reactants were selectively infused via the superior thyroid artery to one thyroid lobe. The contralateral lobe served as a control. In the rat, a supramaximal i.v. dose of thyrotropin (TSH, 500 mU) induced a slight increase in thyroidal tissue levels of cyclic adenosine monophosphate (cAMP) while TSH 50 mU i.v. had no effect on cAMP levels. On the other hand both doses of TSH stimulated exocytosis, signified by a decrease in the number of exocytotic vesicles and endocytosis, signified by the appearance of pseudopods and colloid droplets. Selective thyroid infusion of dbcAMP (5 mM) or forskolin (25 microM), which induced a 10-fold increase in thyroid cAMP levels, did not induce any morphological sign of exocytosis or endocytosis in the follicle cells. The morphological response to TSH given i.v. was quantitatively unaltered by simultaneous infusion of forskolin. In contrast to the findings in rats, infusion of forskolin and dbcAMP in mice induced endocytosis. In conclusion, our findings in the mouse are in agreement with earlier studies in this and other species, indicating that cAMP mediates the effects of TSH on endocytosis and probably also on exocytosis. In contrast, our observations in the rat thyroid in vivo lead to the conclusion that cAMP is not the main intracellular mediator of exocytosis and endocytosis in this species. This conclusion is at variance with previous reports, mostly from in vitro studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Iodine binding and peroxidase activity in the endostyle of Salpa fusiformis, Thalia democratica, Dolioletta gegenbauri and Doliolum nationalis (Tunicata, Thaliacea).

The protothyroid region in the endostyles of four species of tunicates was examined by means of autoradiography and cytochemistry, at both the light and electron-microscopic levels. To reveal the primary binding site for iodine, autoradiography was carried out on endostylar tissue from animals that had been incubated with high activity 125I over a short period of time. The specific iodine binding enzyme, a peroxidase, was traced by its reaction with DAB. In accordance with previous findings, the iodine-binding cells proved to be the same as those containing the peroxidase. There were also strong indications of a secondary uptake of iodinated compounds and subsequent release into the body fluid. Together with the ultrastructural features, the data provided strong evidence indicating that these cells constitute a protothyroid region, which partly functions as an endocrine organ, possibly homologous with the vertebrate thyroid gland. Since the number of zones varied between the species, the numeration of the protothyroid region also varied. However, in all the examined endostyles, the protothyroid region was seen to be situated dorsolaterally to the glandular regions of the endostyle concerned with food capture.

Electron-microscopic immunocytochemistry of 5-hydroxytryptamine in the ascidian endostyle.

The cellular and subcellular distribution of serotonin (5-hydroxytryptamine, 5-HT) in the endostyle of three species of ascidians, Ciona intestinalis, Corella parallelogramma, Ascidia mentula, was studied by light- (immunoperoxidase) and electron-microscopic (immunogold) immunocytochemistry. At the light-microscopic level 5-HT-like immunoreactivity (5-HT-LI) was exclusively found in cells located in the lateral portion of the endostyle, between zone 7, known to have iodinating capacity, and zone 8, which consists of ciliated cells. At the electron-microscopic level, the 5-HT-immunoreactive cells were found to correspond to cells containing polymorphous, dense granules, 100-300 nm in diameter. The granules were located in the supranuclear cytoplasm facing the endostyle lumen as well as in the infranuclear cytoplasm facing the extracellular space. Quantification showed that the 5-HT-LI was considerably higher (13-67 times) in cytoplasmic areas containing granules as compared to areas devoid of granules. Most, but not all, of the 5-HT-LI was associated with the dense core of the granules. In conclusion, serotonin-containing cells are located in the peripheral portion of the endostyle, between zones 7 and 8. Serotonin is stored in cytoplasmic granules that are present both in the apical and basal cytoplasm. This suggests the possibility that the cells are bipolar and secrete serotonin both in a basal direction to the extracellular space, and in an apical direction to the pharyngeal lumen.

Ultrastructural demonstration of iodine binding and peroxidase activity in the endostyle of Oikopleura dioica (Appendicularia).

The endostyles of cephalochordates, ascidians, and larval petromyzontids have the capacity to organify iodine. A similar mechanism in the appendicularian endostyle has hitherto been unknown. Observations in this study of Oikopleura dioica with electron microscopic autoradiography and cytochemistry show that also the appendicularian endostyle has iodinating capacity and that the iodinating cells contain peroxidase, an enzyme responsible for iodination. After incubation in seawater containing 125I-, autoradiography revealed a selective labeling in the dorsal portion of the endostyle. The endostyle of O. dioica is on each side lined by four rows of corridor cells. The autoradiographic grains were mainly located over the endostylar lumen or associated with the luminal surface of the two central rows of corridor cells. These cells, but no other endostylar cells, also showed a positive reaction for peroxidase. The reaction product was distributed along the luminal plasma membrane and was also present in the cytoplasm within rough endoplasmatic reticulum, Golgi apparatus, and vesicles. The selective labeling as well as the cytochemical reaction were abolished by incubation in methimazole, an inhibitor of peroxidase. It is suggested that the two central rows of the corridor cells can be considered as homologs to iodine-binding zones in other endostyles and also as a primitive forerunner to the vertebrate thyroid gland.

Effects of TSH on iodination in rat thyroid follicles studied by autoradiography.

The possible functional relation between TSH-stimulated exocytosis and TSH-stimulated iodination in the thyroid gland was studied using quantitative EM autoradiography and cytochemistry. The study was performed in rats pretreated with thyroxine for 2 days. TSH, giving i.v. 10 min before sacrifice to thyroxine-treated rats, increased organification of 125I by about 50%. TSH decreased the number of peroxidase-positive vesicles in the apical cytoplasm and increased the width of the peroxidase reaction at the apical plasma membrane, suggesting a redistribution of peroxidase. EM autoradiography after labelling with [3H]leucine showed that TSH caused a rapid redistribution by exocytosis of newly synthesized protein to the follicle lumen. The protein deposited in the lumen remained to a large extent in the microvillus region. 10 min after injection of 125I, newly iodinated protein was distributed in a gradient in the lumen periphery. TSH, given 5 min before 125I, caused a significant increase in the labelling of the colloid in the microvillus region, indicating a selective incorporation of iodine into newly synthesized molecules deposited in this region by stimulated exocytosis. Our results confirm and extend earlier observations on a functional link between exocytosis and iodination. Redistribution of peroxidase as well as newly synthesized protein to the site of iodination might be of importance.

Turnover of apical plasma membrane in thyroid follicle cells of normal and thyroxine-treated rats.

In thyroid follicle cells exocytotic vesicles transfer newly synthesized thyroglobulin to the follicle lumen and new membrane to the apical plasma membrane. In a previous study data obtained by quantitative electron microscopy were used to estimate the turnover of the pool of exocytotic vesicles in follicle cells of normal and thyroxine-treated (2 days) rats. In the present study, these kinetic data were combined with stereological measurements to calculate the amount of membrane added to the apical plasma membrane by exocytosis and, indirectly, to estimate the turnover of this membrane. In follicle cells of normal rats the area of the membrane added was about 240 micron2/h (180 micron2/h after correction for stereological overestimation) and in thyroxine-treated rats about 105 micron2/h (corrected 80 micron2/h). These areas corresponded to the addition of 1.2% and 0.7% respectively, of the apical plasma membrane per minute. In each cell the total volume of the exocytotic vesicles emptying their content into the follicle lumen in normal rats was 6.2 micron3/h (corrected 4.7 micron3/h) and in thyroxine-treated rats 3.0 micron3/h (corrected 2.3 micron3/h). Considerations based on the findings in the present study suggest that in normal rats macropinocytosis (formation of colloid droplets) is the most important endocytotic pathway for internalization of colloid, while micropinocytosis is the major pathway of membrane internalization. In thyroxine-treated rats macropinocytosis is inhibited. The low volume capacity of the micropinocytotic pathway probably explains the accumulation of colloid protein, mainly thyroglobulin, observed in such rats.

Rate of protein transport in thyroid follicle cells of normal, thyroxine-treated and TSH-injected rats.

The transport of labeled protein in thyroid follicles was studied with quantitative electron microscopic autoradiography in normal and T4-treated rats (2d) injected with 3H-leucine 1 to 6 h before perfusion fixation. During this time interval the total amount of labeled protein in either group was unchanged, although T4-treatment caused a reduction by about 30% of the amount of 3H-leucine incorporated into protein. The autoradiographic data were corrected for the effect of scatter of radioactivity. The relative amounts of labeled, exportable protein in the compartments Er-Golgi and exocytotic vesicles were then estimated. The half-lives of labeled, exportable protein in these compartments were calculated with non-linear regression analysis. In normal rats the half-life of labeled, exportable protein in ER-Golgi was 28 min and in the exocytotic vesicles 18 min. Inhibition of TSH-secretion by injection of thyroxine decreased the rate of protein transport through the follicle cell and increased the half-lives to 63 min (ER-Golgi) and 62 min (exocytotic vesicles). TSH given to thyroxine-treated rats 20 min or 1.5 h before fixation reduced the half-lives of labeled, exportable protein in ER-Golgi to 25 to 33 min and in exocytotic vesicles to 9 min. The findings indicate that TSH regulates the rate of intracellular protein transport in rat thyroid follicle cells at the exocytotic step as well as at an earlier step in the pathway of intracellular protein transport. The mechanism and exact location of the latter TSH regulated step is at present unknown.

Intraluminal iodination of thyroglobulin.

The intraluminal distribution of newly synthesized (injection of [3H]leucine) and newly iodinated (injection of Na125I) proteins in thyroids of rats given T4 for 2 days was studied with quantitative electron microscopic autoradiography. Three, 4.5, and 6 h after [3H]leucine about 90%, 85%, and 65%, respectively, of the luminal label was confined to the microvillus region. This distribution differed from that of newly iodinated protein; already 2 min after injection only about 30% of the grains was located over the microvillus region. The remaining 70% of the grains located outside the microvillus region formed a gradient towards the center of the lumen. The grain distributions 30 min and 2 h after Na125I were similar to that present after 2 min. The distribution of grains after pulse labeling with Na125I (injected 2 min before propylthiouracil and 2 h before fixation) was also similar to that found in rats injected with Na125I alone, indicating that diffusion of labeled proteins in the lumen was very slow in T4-treated rats. A slow diffusion was also suggested by the presence of an unlabeled peripheral ring in follicle lumens of T4-treated rats injected with Na125I 48 h before fixation. In normal rats given [3H]leucine 3 h before fixation or Na125I 1 h or 48 h before fixation the grains were homogeneously distributed in most follicle lumens. Together our findings indicate that (1) administration of T4 has effects on the diffusion properties of the colloid; (2) iodine is incorporated not only into newly synthesized thyroglobulin recently delivered to the follicle lumen but also into molecules already stored in the lumen; (3) a portion of the iodine incorporated into proteins is bound to molecules which are not in direct contact with thyroperoxidase in the apical plasma membrane.

Diffusion artefacts and tissue fixation in thyroperoxidase cytochemistry.

The distribution of endogenous peroxidase activity in rat, mouse and human thyroid follicle cells was studied with electron microscopic cytochemistry after incubation in 3-3'-diaminobenzidine (DAB). In all three species enzyme activity was found at the apical plasma membrane (facing the follicle lumen) as well as in intracellular compartments. The enzyme activity in the apical plasma membrane was more sensitive to changes in fixation conditions than the activity in intracellular compartments. Under optimal conditions more than 90% of the follicle cells in normal rat thyroids displayed a cytochemical reaction at the apical plasma membrane. In all three species the reaction product at the apical plasma membrane formed a gradient which extended into the colloid which otherwise was unreactive. Evidence obtained indicated that this gradient was not due to the presence of soluble peroxidase in the lumen but most likely signified the diffusion of the reaction product from the membrane-bound enzyme.

In vivo shedding of apical plasma membrane in the thyroid follicle cells of the mouse.

Clusters of luminal dense bodies, limited by a triple-layered membrane, were found in all follicle lumina in thyroid glands of mice. After thyroxine treatment the number of luminal dense bodies increased, especially in the periphery of the lumen, where the intraluminal bodies often displayed a striking resemblance to microvilli. In hyperplastic goiters, obtained by feeding mice with propylthiouracil, luminal dense bodies were replaced by intraluminal vesicles. During goiter involution the vesicles were gradually replaced by luminal dense bodies; the presence of intermediate forms suggests that vesicles and dense bodies are basically the same formations. Luminal dense bodies were observed in colloid droplets indicating their removal by endocytosis. As demonstrated by electron-microscopic cytochemistry, luminal dense bodies contain a membrane-bound peroxidase, and electron-microscopic autoradiography after administration of 125I indicate that they possess an iodinating capacity. Our observations on mouse thyroid glands suggest that the luminal dense bodies, which appear as vesicles in hyperplastic glands, are formed by shedding of the apical plasma membrane of the follicle cell. The shedding process might be of importance for the turnover of plasma-membrane material.

Selective macropinocytosis of thyroglobulin in rat thyroid follicles.

We have explored the possibility that pseudopods might provide a mechanism by which newly synthesized, hormone-poor thyroglobulin recently delivered to the follicle lumen escapes immediate reuptake and degradation. The study was performed with electron microscopic autoradiography on rats pretreated with T4 for 2 days and injected with [3H]leucine or Na125I. Pseudopods were induced by the injection of TSH (100 mU) 20 min before perfusion fixation. The density of autoradiographic grains in colloid droplets located in pseudopods was compared with the grain density in different regions of the follicle lumen. In rats injected with radioleucine 1.5 or 3 h before TSH injection, the grains were distributed in a gradient in the lumen periphery. Seventy to 80% of the grains were located over the microvillus region. The grain density over colloid droplets in pseudopods was about 10% of that over the microvillus region and similar to the density at a distance 1-2 microns from the microvillus region. After injection of Na125I, 40 min before TSH, the grains were more widely spread in the lumen, but still formed a gradient in the lumen; about 30% of the grains were associated with the microvillus region. Again, the grain density over colloid droplets in pseudopods was about the same as that at a distance 1-2 microns from the microvillus region. Our observations are compatible with the idea that pseudopods collect thyroglobulin located at some distance from the apical surface. This, together with the circumstance that newly synthesized thyroglobulin is located close to the apical plasma membrane, might provide a mechanism of selective macropinocytosis by which newly synthesized thyroglobulin recently delivered to the follicle lumen is prevented from immediate reuptake.

Transport of thyroglobulin and peroxidase in the thyroid follicle cell.

The purpose of this study was to explore the nature of the protein(s) in the exocytotic vesicles in the thyroid follicle cells and to ascertain whether or not thyroglobulin and peroxidase are transported by the same vesicles through the apical region of the cells to the follicle lumen. The study was performed on rats pretreated with thyroxine for 2 days in order to inhibit endocytosis. A fraction of exocytotic vesicles was isolated by centrifugation in continuous and discontinuous sucrose density gradients. The protein content of the vesicles were analysed by electrophoresis in continuous polyacrylamide gradient gels. The vesicles contained (uniodinated) thyroglobulin, 12-S protein and thyralbumin. Parallel histochemical studies in the electron microscope. These observations have important bearings on the mechanisms for thyroglobulin iodination, since it has been demonstrated that iodination does not occur in the exocytotic vesicles but in connection with the opening of the vesicles at the apical cell surface.