UPTAKE OF L-TRI-IODOTHYRONINE BY ISOLATED RAT-LIVER CELLS - PROCESS PARTIALLY INHIBITED BY METABOLIC-INHIBITORS - ATTEMPTS TO DISTINGUISH BETWEEN UPTAKE AND BINDING TO INTRACELLULAR PROTEINS

Rat liver cells obtained by dispersion with collagenase were used to investigate the mode of entry of L-tri-iodothyronine into the cell. The hormone was taken up very rapidly at 23°C; the linear phase of uptake lasted for up to approx. 20s. 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±15pm was designated as system I, and the second uptake component with an apparent Kt of 726±11pm 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. Uptake of L-tri-iodothyronine by system I was higher at pH6.4 than at pH7.4; system II was relatively insensitive to changes in the pH of the external medium. Both systems exhibited a transition temperature at about 16°C in the Arrhenius plot. The activation energies of the two systems below and above 16°C were 72.8 and 47.7, and 54.4 and 33.1J/mol, respectively. Inhibitors of cellular energy reduced the uptake by system I to a larger extent than that by system II. Replacement of Na+ in the external medium by either K+ or choline led to uptake that followed normal Michaelis-Menten kinetics. Thiol-group-blocking agents reduced the uptake of the hormone by both systems. Treatment of liver cells with β-glucosidase, Pronase neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I, whereas by system II was decreased after treatment with phospholipase A2, β-galactosidase, Pronase and neuraminidase. The stereoisomer D-tri-iodothyronine (100-3000pM) did not affect system I, but uptake by system II decreased with increasing concentration of D-tri-iodothyronine. Reverse L-tri-iodothyronine (2-100pM) and L-thyroxine (100-3000pM) did not influence uptake by either system. 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 result suggest that cytosol proteins are not directly involved in the uptake of L-tri-iodothyronine. Plasma-membrane vesicles also take up the hormone rapidly at 23°C. Increasing the osmolarity of the external medium led to a decrease in the uptake of L-tri-iodothyronine by vesicles. Uptake as a function of L-tri-iodothyronine concentration exhibited a sigmoidal cruve. the Eadie-Hofstee plot showed two uptake components with apparent Kt values of 96.8 and 1581 pM. The results of our study are consistent with a carrier-mediated translocation of the hormone into the cell.