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Lars E. Ericson and Ragnar Ekholm

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Lars E Ericson and Mikael Nilsson

Ericson LE, Nilsson M. Effects of insulin-like growth factor I on growth, epithelial barrier and iodide transport in polarized pig thyrocyte monolayers. Eur J Endocrinol 1996;135:118–27. ISSN 0804–4643

The effects of insulin-like growth factor I (IGF-I) on mitogenesis, epithelial barrier function and transepithelial iodide transport were studied in confluent, polarized monolayers of pig thyrocytes cultured on filter in Transwell bicameral chambers. The growth rate in controls cultured in 1% fetal calf serum was low. Insulin-like growth factor I stimulated dose-dependently the incorporation of [3H]thymidine, maximally at 100 ng/ml, which corresponded to an increase of DNA content by 60% after 6 days. Thyrotropin (1 mU/ml) alone did not stimulate cell multiplication but inhibited partially the stimulatory effect of IGF-I. Insulin-like growth factor I(100 ng/ml) increased within 10 min the transepithelial potential difference, which remained elevated for several days, but did not significantly change the transepithelial resistance. When added together, IGF-I reinforced the effects of TSH on potential difference (increase) and resistance (decrease). A preserved epithelial barrier in IGF-I-treated cultures was confirmed by observing a normal immunolocalization of the tight junction protein ZO-1 and an unchanged ultrastructure of the junctional complex. Insulin-like growth factor I increased the transepithelial flux of 125I in the basal-to-apical, but not in the opposite, direction. Stimulation of iodide transport by IGF-I was modest after 2 days and pronounced after 6 days. In comparison, TSH-stimulated iodide transport was higher after 2 days but lower after 6 days. Both TSH and IGF-I were strongly synergistic, after 6 days amounting to a 90-fold increase over the control basoapical 125I transfer. The simultaneous accumulation of 125I in the cell layer was increased two- to fourfold by IGF-I and/or TSH. In conclusion, IGF-I is able to induce growth in preformed monolayers of pig thyrocytes cultured on permeable filter. During these conditions, the mitogenic effect of IGF-I is partially inhibited by TSH, which has no growth-promoting action on its own. The transepithelial transport of iodide and bulk electrolytes is altered by IGF-I without affecting the epithelial barrier function. Specifically, IGF-I up-regulates the activity of the basolateral iodide pump and increases the iodide permeability of the apical plasma membrane. The action of IGF-I on iodide transport is independent of, although synergistic with, that of TSH. The findings support the notion that IGF-I may be an important regulator of thyroid growth and differentiated functions.

Lars E Ericson, Institute of Anatomy and Cell Biology, Göteborg University, Medicinaregatan 3, S-413 90 Göteborg, Sweden

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Ingmar Lundquist, Ragnar Ekholm and Lars E. Ericson

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Viktor Johanson, Torsten Ofverholm and Lars E. Ericson

Abstract. 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 sustances are unsuitable. The technique permits selective administration of small amounts of experimental substances to the thyroid in high concentrations.

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Lars E. Ericson and Bengt R. Johansson

ABSTRACT

The early effect (3 min) of thyroid stimulating hormone (TSH) on exocytosis-endocytosis in the rat thyroid follicle cell was investigated by electron microscopic morphometry. All rats used were pre-treated with thyroxine for 2 days prior to the experiments. In control rats, the membrane surface area of exocytotic vesicles, the only type of vesicle present in the apical part of the follicle cell, approximately equaled that of the apical plasma membrane. Intravenous administration of TSH caused a significant decrease in the membrane surface area of exocytotic vesicles by about 20 % and a corresponding, significant increase in the membrane surface area of the apical plasma membrane by about 20 %. Pseudopods were present in 4 of 6 TSH-injected rats examined, but the membrane surface area of these structures was only 15 % of the increase in the surface area of the apical plasma membrane. The total membrane surface area analyzed was not influenced by injection of TSH.

We conclude that the initial effect of TSH on the thyroid follicle cell is a re-distribution of membrane from exocytotic vesicles to the apical plasma membrane. This conclusion is in harmony with our previous observations which indicate that the (membrane-requiring) endocytotic response after TSH is regulated by the amount of membrane material added to the apical plasma membrane by exocytosis.

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Mikael Nilsson, Ulla Björkman, Ragnar Ekholm and Lars E Ericson

The intracellular regulation of thyrotropin-stimulated iodide efflux was studied in polarized porcine thyrocytes grown as a continuous, tight monolayer in bicameral culture chambers. From a previous study using this system we know that thyrotropin rapidly increases iodide efflux in the apical but not basal direction of the polarized epithelium. [125I]-iodide efflux in apical direction was stimulated by thyrotropin in a concentration-dependent manner (1–10 U/I), whereas efflux in basal direction was unchanged at any thyrotropin dose. Thyrotropin-induced elevation of intracellular cAMP showed a corresponding concentration dependence. The selective stimulation of apical efflux by thyrotropin was evident also when re-uptake of iodide released in basal direction was blocked by perchlorate. The effect of thyrotropin on apical efflux was mimicked by 8-bromo-cAMP and forskolin, whereas agents known to activate the Ca2+/phosphatidylinositol cascade (epidermal growth factor) and protein kinase C (phorbol ester) or increase cytosolic [Ca2+] (A23187) were inactive. We conclude that the selective stimulation by thyrotropin of apical iodide efflux, corresponding to efflux in luminal direction in intact follicles, occurs via cAMP-regulated iodide channels present in the apical domain of the plasma membrane.

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Mikael Nilsson, Johanna Husmark, Bengt Nilsson, Lars-Erik Tisell and Lars E Ericson

Nilsson M, Husmark J, Nilsson B, Tisell L-E, Ericson LE. Primary culture of human thyrocytes in Transwell bicameral chamber: thyrotropin promotes polarization and epithelial barrier function. Eur J Endocrinol 1996;135:469–80. ISSN 0804–4643

Epithelial properties of thyrocytes are difficult to maintain in conventional cell culture systems. We used bicameral chambers (Transwell™) in attempts to establish a functional epithelium of thyrocytes of human origin. Thyroid follicle segments were isolated by collagenase digestion of paradenomatous tissue obtained at surgery for follicular adenoma and of tissue from glands with Graves' disease. After careful separation from connective tissue and single cells by centrifugation, the follicles were plated at high density on the collagen-coated filter of the chambers and cultured in Eagle's essential medium (EMEM) containing 10% fetal calf serum (FCS) or Coon's modified Hams medium enriched with five or six factors (5H, 6H); the latter media contained 5% FCS without (5H) or with (6H) thyrotropin (TSH). The follicles were converted into a confluent cell layer, which had similar DNA content irrespective of type of medium, after 4–6 days. Cells grown in EMEM or 5H established a transepithelial electrical resistance (R) of 200–500Ω·cm2 and was impermeable to [3H]inulin, indicating the formation of epithelial junctions. Addition of 6H to confluent cells initially cultured in EMEM or 5H caused a further increase of R, maximally to 1500 Ω·cm2, along with a rise of the transepithelial potential difference; 6H promoted the monolayer formation of cells, increased the number of apical microvilli and reinforced the junctional distribution of actin, cadherin and ZO-1; 6H also enhanced the polarized secretion of [3H]leucine-labeled thyroglobulin into the apical medium. Cells from Graves' thyroid tissue established an epithelium on the filter with similar characteristics to that of normal thyrocytes; some platings contained in addition large numbers of HLA-DR positive cells with a dendritic shape. HLA-DR expression was generally absent in EMEM- or 5H-grown thyrocytes, but appeared in limited areas of the cell layer after 6H and was expressed by all epithelial cells after interferon-gamma stimulation for 48 h. We conclude that human thyrocytes form a tight and polarized epithelium when cultured on permeable filters. The polarized structure and function of the cells are positively regulated by TSH. The culture system may be useful in studies addressing the role of the epithelial phenotype (cell polarity and tight barrier) in normal thyroid function as well as in pathological processes in the thyroid, such as autoimmunity, cell transformation and tumor progression.

Mikael Nilsson, Institute of Anatomy and Cell Biology, Göteborg University, Medicinaregatan 3, S-413 90 Göteborg, Sweden

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Bengt Hallengren, Mervyn P. Hegarty, Arne Forsgren, Lars E. Ericson and Arne Melander

Abstract. 3,4-Dihydroxypyridine (3,4-DHP), a goitrogenic derivative of the plant amino acid mimosine, has no SH-group, in contrast to conventional antithyroid agents such as methimazole (MMI) and propylthiouracil (PTU). The current in vitro study shows that 3,4-DHP, like MMI and PTU, inhibits iodination of human thyroglobulin and interferes with mitogenic activation of human lymphocytes. This, together with a very low murine bone marrow toxicity, probably related to the absence of an SH-group, makes 3,4-DHP a potential antithyroid drug.