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

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.degree. C; the linear phase of uptake lasted for up to .apprx. 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 vs. v) yielded 2 straight lines. The uptake component with an apparent Kt value of 86 .+-. 15 pM was designated as system I, and the 2nd 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 2.1. 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. Both systems exhibited a transition temperature at about 16.degree. C in the Arrhenius plot. The activation energies of the 2 systems below and above 16.degree. C were 72.8 and 47.7, and 54.4 and 33.1 J/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 .beta.-glucosidase, pronase and neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I; uptake by system II was decreased after treatment with phospholipase A2, .beta.-galactosidase, pronase and neuraminidase. 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. 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. Cytosol proteins apparently are not directly involved in the uptake of L-tri-iodothyronine. Plasma-membrane vesicles also take up the hormone rapidly at 23.degree. 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 curve. The Eadie-Hofstee plot showed two uptake components with apparent Kt values of 96.8 and 1581 pM. A carrier-mediated translocation of the hormone into the cell is supported.