Uptake of L-tri-iodothyronine by isolated rat liver cells. A process partially inhibited by metabolic inhibitors; attempts to distinguish between uptake and binding to intracellular proteins

Abstract

1. Rat liver cells obtained by dispersion with collagenase were used to investigate the mode of entry of L-tri-iodothyronine into the cell. 2. The hormone was taken up very rapidly at 23 degrees C; the linear phase of uptake lasted for up to approx. 20 s. 3. A plot of the initial rates of uptake against different concentrations of L-tri-iodothyronine yielded a sigmoidal curve. The Eadie--Hofstee plot (v/[S]2 versus v) yielded two straight lines. The uptake component with an apparent Kt value of 86 +/- 15 pM was designated as system I, and the second uptake component with an apparent Kt of 726 +/- 11 pM as system II. The Hill plot for system I was not linear; the apparent Hill coefficient for system II was calculated to be 2.1.4. Uptake of L-tri-iodothyronine by system I was higher at pH 6.4 than at pH 7.4; system II was relatively insensitive to changes in the pH of the external medium. 5. Both systems exhibited a transition temperature at about 16 degrees C in the Arrhenius plot. The activation energies of the two systems below and above 16 degrees C were 72.8 and 47.7 and 54.4 and 33.1 J/mol respectively. 6. Inhibitors of cellular energy reduced the uptake by system I to a larger extent than that by system II. 7. Replacement of Na+ in the external medium by either K+ or choline led to uptake that followed normal Michaelis--Menten kinetics. 8. Thiol-group-blocking agents reduced the uptake of the hormone by both systems. 9. Treatment of liver cells with beta-glucosidase, Pronase and neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I, whereas uptake by system II was decreased after treatment with phospholipase A2, beta-galactosidase. Pronase and neuraminidase. 10. The stereoisomer D-tri-iodothyronine (100--3000 pM) did not affect system I, but uptake by system II decreased with increasing concentration of D-tri-iodothyronine. Reverse L-tri-iodothyronine (2--100 pM) and L-thyroxine (100--3000 pM) did not influence uptake by either system. 11. Under identical conditions of incubation, the uptake of L-tri-iodothyronine was 3.7 times higher than binding to cytosol proteins. The binding was insensitive to metabolic inhibitors. The results suggest that cytosol proteins are not directly involved in the uptake of L-tri-iodothyronine. 12. Plasma-membrane vesicles also take up the hormone rapidly at 23 degrees C. Increasing the osmolarity of the external medium led to a decrease in the uptake of L-tri-iodothyronine by vesicles. 13. Uptake as a function of L-tri-iodothyronine concentration exhibited a sigmoidal curve. The Eadie--Hofstee plot showed two uptake components with apparent Kt values of 96.8 and 1581 pM. 14. The results of our study are consistent with a carrier-mediated translocation of the hormone into the cell.

Influx of Thyroid Hormones into Rat Liver In Vivo

“…The transport of T3 into isolated liver cells at 21-23°C has been shown to be saturable (22,23). A similar discrepancy has been observed for liver cell membrane transport of the steroid hormones, wherein steroid transport in vivo is nonsaturable (7), but hormone transport in vitro is saturable (24).…”

“…Although the high lipid solubility of T4 and T3 and the nonsaturability of thyronine transport into liver is evidence in favor of lipid-mediation, other studies reThyroxine and Triiodothyronine Transport into Liver 371 port data in favor of carrier-mediation (22,23). The transport of T3 into isolated liver cells at 21-23°C has been shown to be saturable (22,23).…”

Receptor-mediated uptake of 3,3',5-triiodo-L-thyronine by cultured fibroblasts.

“…Our The presence of saturable and specific binding components for thyroid hormone in purified rat liver plasma membrane was reported by Pliam and Goldfine (7), although the physiological role of these T3 receptors was not defined. Entry of T3 into cells by a receptor-mediated process was suggested by others, using isolated rat liver cells (10,11,12), human erythrocyte ghosts (8), and rabbit adipocytes (9). However, the mechanism of the uptake was not elucidated.…”

“…and coworkers (11,12) independently, the mechanism of receptor-mediated uptake has not yet been elucidated. To study this question, we synthesized tetramethylrhodamine (Rho)-labeled T3 and showed that it possessed biological activity (13).…”

Affinity labeling of the plasma membrane 3,3',5-triiodo-L-thyronine receptor in GH3 cells.

“…Over the past two decades it has been believed that the delivery of thyroid hormones into cells was by simple passive diffusion (2)(3)(4). However, recent evidence indicates that the uptake of thyroid hormones into cells occurs by a receptor-mediated process (5)(6)(7)(8)(9)(10)(11)(12). We have shown that the uptake of3,3',5-triiodo-L-thyronine (T3) into GH3 rat pituitary tumor cells occurs by receptor-mediated endocytosis and that this mode of entry is physiologically significant (13).…”