THYROTROPIN CHRONICALLY REGULATES THE POOL OF THYROPEROXIDASE AND ITSINTRACELLULAR-DISTRIBUTION - A QUANTITATIVE CONFOCAL MICROSCOPIC STUDY

The regulation of thyroperoxidase (TPO) expression and of its intracel lular distribution was studied in porcine thyroid cells cultured on po rous bottom filters. Cells were cultured for 18 days in the absence or in the presence of thyrotropin (TSH) and with or without iodide. Micr osomes were purified and analyzed by electrophoresis. TPO was detected by immunoblotting with polyclonal anti-porcine TPO antibodies and qua ntified by scanning the bands. The amount of TPO was increased 2-fold by TSH. High concentrations of iodide (1-50 mu M, added daily) decreas ed the level of TPO. Confocal microscopy served to determine the intra cellular localization of TPO and its quantitative distribution. Intrac ellular and surface-located TPO was detected by fluorescein-labeled an tibodies on saponin-treated cells. Quantitative confocal microscopy sh owed that TSH increased the total amount of TPO 2-fold as for immunobl otting. The highest amount of TPO was found in the perinuclear area an d between the nucleus and the Golgi apparatus. Only 4% of TPO was pres ent on the apical surface and about 1% on the basolateral membrane; th e remainder (about 95%) was inside the cells. TSH did not change these relative contents. TSH modified the intracellular distribution of the enzyme, increasing the TPO pool from the perinuclear area to apical m embrane. This domain could be a site of storage of TPO. Adding a physi ological concentration of iodide (0.5 mu M, daily) did not influence t he intracellular distribution of TPO. We concluded that chronic TSH st imulation 1) increased 2-fold the pool of TPO but did not change the r elative proportion of TPO inside the cells and on the apical surface, and 2) modified the intracellular distribution of vesicular TPO, the m ajor part of which was accumulated in the perinuclear and cytoplasmic area under the subapical domain of the polarized cells. (C) 1998 Wiley -Liss, Inc.