Synthesis and apical and basolateral secretion of thyroglobulin by thyroid cell monolayers on permeable substrate: Modulation by thyrotropin

Abstract

In the thyroid glands, thyroglobulin (Tg) is specifically synthesized by follicular cells and then secreted into the apical lumen where it is concentrated and used as a substrate for thyroid hormone synthesis. The presence of Tg in the circulation has been reported in normal and pathological situations. To determine the domains of the plasma membrane, apical and/or basolateral, involved in Tg secretion, porcine thyroid epithelial cells were cultured as a monolayer on the porous bottom of a culture chamber in which both apical and basal media are independently accessible. Control experiments using labeled Tg ascertained the tightness of the monolayer and showed that within 48 h only 0.2-0.5% of the Tg introduced in the apical medium was transferred through the cell layer into the basal compartment. For kinetic studies of Tg synthesis and secretion, monolayers were cultured for up to 72 h in the presence of 35S-methionine and with or without 100 microU/ml thyrotropin (TSH) in the basal medium. Labeled Tg was measured by double immunoprecipitation and by fluorography of polyacrylamide gel electrophoresis. We showed that 80-95% of total secreted Tg was recovered in the apical medium. The remainder was secreted through the basolateral membranes in the basal medium. The amount of tg secreted into the apical compartment was stimulated two- to threefold by TSH whereas no TSH effect was observed on secretion in the basal compartment. Moreover, measuring apical and basal volumes, we observed a net water flow from the apical to the basal side. It was stimulated threefold by TSH, increasing the Tg concentration in the apical compartment of the stimulated cell layer. During the culture time, the amount of Tg synthesized and secreted was increased by TSH, as was the Tg mRNA content, as determined by the dot-blot hybridization method.

Small conductance chloride channels in the apical membrane of thyroid cells

“…When thyroid cells are cultured on a solid support, they form a well-polarized monolayer with the basolateral membrane facing the attachment substrate and the apical membrane facing the bath medium [7,8].…”

Thyrotropin regulates tyrosine sulfation of thyroglobulin

“…Epithelial cells were isolated from porcine thyroid glands and cultured on collagen-coated filters as described previously (25). Briefly, isolated cells suspended in phenol red-free Dulbecco's modified Eagle's medium (DMEM containing glucose 1 g/l, Gibco Brl, Cergy Pontoise, France) were seeded into the apical compartment of chambers.…”

“…From day 4, the basal medium was supplemented with 10% newborn calf serum (Biological Industries, Beth Haemek, Israel), when media were changed. On day 6, the tightness of the monolayer was examined (25) and only tight monolayers were kept. From day 6, 100 mU/ml TSH (b-TSH, Calbiochem, San Diego, CA, USA) was added or not to the basal medium.…”

“…Over many years, we have accumulated extensive data on this culture system, which simulates thyroid metabolism in vivo, up to the stage of secretion of thyroid hormones into the basal medium (unpublished results). We have shown that TSH controlled all the steps of thyroglobulin metabolism that we have studied: thyroglobulin expression and secretion (25)(26)(27)(28), and two post-translational modifications, glycosylation (29,30) and iodination (31).…”

Production of lipocortin‐like proteins by cultured human tracheal submucosal gland cells

“…The tightness of confluent ceils was assessed by means of the technique described by Chambard et al [25]. Ultroser G-free RPMI 1640 medium was added to the apical side while 2070 Ultroser G-supplemented medium was added to the basal side.…”