THYROTROPIN STIMULATION OF THYROXINE SYNTHESIS IN ISOLATED THYROID CELLS TREATED WITH PUROMYCIN.

The in vitro incorporation of I into thyroxine and its precursors, monoiodoand diiodotyrosine, was observed in isolated bovine thyroid cells. This utilization of I was stimulated 2to 3-fold when the cells were incubated in the presence of 0.1 U of TSH/ml. Perchlorate ion added at a concentration of 2 mM completely eliminated iodide-concentrating activity by the cells, but did not significantly impair the stimulatory action of TSH on the incorporation of I into thyroxine and the iodotyrosines. Enhanced iodide-concentrating activity appears not to be required for this action of TSH on thyroxine formation. (Endocrinology 75: 968, 1964) A PRONOUNCED stimulatory action of J\. TSH in vitro on the incorporation of I into T4 and into iodotyrosines by isolated thyroid cells has recently been described (1, 2). This enhanced utilization of I was observed without a concomitant increase in iodide-concentrating activity as reflected by intracellular to extracellular I-iodide concentration (C/M) ratios. In fact, C/M ratios and the rate of iodide accumulation were consistently depressed by added TSH (2). A similar dissociation of the effects of TSH on iodide concentration and iodoprotein formation has been demonstrated in hypophysectomized rats by Halmi et at. (3). Four hr after injection of TSH, thyroidal uptake (and presumably organic binding) of I was increased to more than twice that of control hypophysectomized rats. At the same time, thyroid to serum I-iodide concentration ratios (observed in hypophysectomized rats injected with propylthiouracil) were depressed from a mean control value of 5 to a mean value of 2.1 by the same TSH treatment. From the results of our in vitro experiments with thyroid cells, and Halmi's in vivo observations, it may be inferred that TSH has a Received May 25,1964. Aided by a Research Career Development Award and Research Grant AM-06439 from the USPHS. 1 Abbreviations: TSH for thyroid-stimulating hormone, T4 for thyroxine, MIT for 3-iodo-Ltyrosine and DIT for 3,5-diiodo-L-tyrosine. direct stimulatory action on thyroxine formation that is not dependent upon increased intracellular concentration of iodide. Further support for this conclusion is provided in the present studies in which TSH stimulation of thyroxine (and iodotyrosine) formation was observed in thyroid cells treated with perchlorate to eliminate iodide-concentrating activity. Materials and Methods Procedures for the isolation, incubation and chromatographic analysis of thyroid cells have been described previously (1, 2, 4). For each experiment 140 g of minced bovine thyroid tissue was trypsinized to yield approximately 3 ml of cells. The cells were suspended in 25 volumes of Earle's balanced salt solution, and 5 ml aliquots of this homogeneous cell suspension were distributed into 25 ml Erlenmeyer flasks. In these experiments, all the specimens were preincubated for 15 or 30 min in the presence of 2 mM perchlorate. Then carrier iodide (4X10~M), I-iodide (5 /xc/ml) and TSH (where appropriate) were added and the incubations were continued for 3 hr. After incubation, the cells were recovered by centrifugation for 5 min at 50 X g, homogenized in 1 or 2 ml of a reagent consisting of 0.5% (w/v) of Pronase (Calbiochem), 30 mM Tris (pH 7.5), 2 mM MnS04 and 2 mM 1-methyl, 2-mercaptoimidazole (Tapazole), and hydrolyzed at 37 C for 8 hr. 200 /J aliquots of the resulting hydrolysates were chromatographed on Whatman No. 3 MM paper with a collidine-3N NH40H (100:37) solvent system. The iodide-concentrating activity of each cell preparation was observed and expressed in terms of the ratio of intracellular to extracellular I-