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Open thyroid follicles were prepared by mechanical disruption of pig thyroid fragments through a metal sieve. This procedure allowed preparation of thyroid-cell material depleted of colloid thyroglobulin. Open thyroid follicles were used to prepared a crude particulate fraction, which contained lysosomes, mitochondria and endoplasmic reticulum. These organelles were subfractionated by isopycnic centrifugation on iso-osmotic Percoll gradients. (1) A lysosomal peak was identified by its content of acid hydrolases: acid phosphatase, cathepsin D, ,l-galactosidase and ,-glucuronidase. The lysosomal peak was well separated from mitochondria and endoplasmic reticulum. (2) The lysosomal peak, from which Percoll was removed by centrifugation, was taken as the purified lysosome fraction (L). Lysosomes of fraction L were purified 45-55-fold (as compared with the homogenate) and contained about 5% of the total thyroid acid hydrolase activities. (3) Electron microscopy showed that fraction L was composed of an approx. 90% pure population of lysosomes, with an average diameter of 220 nm. (4) Acid hydrolase activities were almost completely (80-90% ) released by an osmotic-pressure-dependent lysis. (5) Thyroglobulin was identified by polyacrylamide-gel electrophoresis as a soluble component of the lysosome fraction. In conclusion, a 50-fold purification of pig thyroid lysosomes was achieved by using a new tissue-disruption procedure and isopycnic centrifugation on Percoll gradient. The presence of thyroglobulin indicates that the lysosome population is probably composed of primary and secondary lysosomes. Isolated thyroid lysosomes should serve as an interesting model to study the reactions whereby thyroid hormones are generated from thyroglobulin and released into the thyroid cells.

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By microinjection of Lucifer yellow (LY) and analysis of the cell to cell transfer of the fluorescent probe, we have examined 1) the ability of thyroid cells in primary culture to reconstitute gap junctions and 2) the effects of extracellular signals on the functional activity of these junctions. Isolated thyrocytes cultured in tissue culture-treated petri dishes either formed monolayers or reorganized in follicular structures in the presence of the glycoprotein hormone TSH. In both culture conditions, LY-coupled cells were evident after 24-36 h. The communication between cells forming a reconstituted thyroid follicle was maintained for up to 9 days. In contrast, the dye coupling between cells in monolayer progressively decreased with time. The cell to cell communication, i.e., the number of dye-coupled cells in thyroid cell monolayer, was increased by TSH in a time- and concentration-dependent manner. The TSH action was not related to de novo protein synthesis. (Bu)2cAMP exhibited stimulatory effects similar, in terms of time course and amplitude of action, to those of TSH. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate rapidly inhibited both basal and TSH- or (Bu)2 cAMP-activated cell to cell communication. The dye coupling of cells in reconstituted follicles was also blocked by a short 12-O-tetradecanoyl phorbol 13-acetate treatment in both the presence and absence of TSH. Our data show that thyroid cells in culture, regardless of the full expression of the differentiated phenotype, rapidly reestablish intercellular gap junctions. The functional activity of gap junctions appears to be regulated 1) positively by a hormone, TSH, probably acting via the cAMP and protein kinase-A pathway, and 2) negatively by phorbol esters through the activation of protein kinase-C, the two regulatory pathways being interdependent.