STIMULATORY EFFECT OF THYROTROPIN ON SYNTHESIS OF THYROXINE BY ISOLATED THYROID CELLS.

Iodide transport and thyroxine synthesis were studied in isolated cells prepared by trypsinization of bovine thyroid glands. When the cells were incubated in media containing 0.2 unit of thyrotropin (TSH) per ml, incorporation of I into thyroxine (and iodotyrosines) was stimulated 2to 4-fold, while no enhancement of iodideconcentrating activity could be detected. (Endocrinology 74: 304, 1964) D cells, obtained from ovine thyroid tissue by trypsinization, have been shown to retain the capacity to concentrate iodide, and to incorporate I-labeled iodide into iodotyrosines and iodothyronines (1). In the present study, the effects of added thyrotropin on these functions of the isolated cell preparation were investigated. Materials and Methods Dispersed thyroid cells were prepared by means of the continuous-flow trypsinization technique described previously (1). The following modifications of the technique were used in the present study: (a) bovine thyroid tissue was used instead of ovine tissue, (b) Earle's balanced salt solution was used in place of Hank's solution to prepare the trypsinizing reagent, (c) the freshly collected cells were washed with a dilute DNase solution to eliminate "sliminess" so that homogeneous cell suspensions could be obtained. In each experiment 2-3 ml of a fresh thyroid cell preparation was suspended in 50 ml of Earle's solution containing 4XlO~ M Nal labeled with I. 5 ml aliquots of such cell suspensions, containing 0.2-0.3 ml of cells, were delivered into 25 ml Erlenmeyer flasks and incubated under 95 % O2-5 % CO2 with shaking (90 oscillations/min) at 37 C in a Dubnoff water bath. After incubation, the contents of the flasks were transferred to Shevky-Stafford type centrifuge tubes, and the cells were sedimented by centrifugation for 5 min at 50 Xg in an International centrifuge maintained at a temperature of 5 C. Packed cell volumes were read to the nearest 0.01 ml, and then the supernatant media were drawn off. Iodine-concentrating activity was expressed as C/M ratios, I-iodide per ml cells I-iodide per ml medium The formation of organically bound I was prevented by incubating in the presence of 0.002 M Tapazole, so that I assays performed on packed cell pellets and aliquots of incubating media provided a direct measure of I-iodide distribution. However, the C/M values presented below are not corrected for the volume of the media trapped in the packed cell pellets. Extracellular space determined with C-inulin and I-albumin amounted to 49-60% of the packed cell volumes; hence, the true intracellular I-iodide concentrations were probably about twice those indicated by the uncorrected C/M values. In the chromatographic studies, I-labeled cells were homogenized in 10 volumes of 0.03 M tris buffer solution (pH 7.5) which contained 0.5 % (w/v) of Pronase. After incubation for 6-9 hr at 37 C, 200 /J aliquots of the hydrolysates were chromatographed on Whatman No. 3 MM paper in collidine-3N NH40H (1). r e labeled components were located by radioautography and assayed by means of a well-type Nal crystal, scintillation detector. Received September 4, 1963. Aided by USPHS Grant AM-06439. 1 Worthington desoxyribonuclease I. 2 Purchased from Fisher Scientific Co. 3 Abbreviations: Tapazole for 1-methyl, 2mercaptoimidazole; TSH for thyroid-stimulating hormone; MIT for 3-iodotyrosine; DIT for 3,5-diiodotyrosine; T4 for thyroxine; and tris for tris (hydroxymethyl) amino methane. 4 Pronase, Streptomyces griseus protease, was obtained from California Corporation for Biochemical Research.