Synthesis and apical and basolateral secretion of thyroglobulin by thyroid cell monolayers on permeable substrate: modulation by thyrotropin.
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.
Chambard
M
M
INSERM U 270 Faculté de Médecine, Secteur Nord, Marseille, France.
Mauchamp
J
J
Chabaud
O
O
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D001798:Blood Proteins / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D008715:Methionine / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000032:analysis; D013552:Swine; D013954:Thyroglobulin / Q000235:genetics / Q000378:metabolism*; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism*; D013972:Thyrotropin / Q000494:pharmacology*
D001798:Blood Proteins; D012333:RNA, Messenger; D013972:Thyrotropin; D013954:Thyroglobulin; D008715:Methionine