Evidence that the uptake of tri-iodo-l-thyronine by human erythrocytes is carrier-mediated but not energy-dependent

We investigated 3,3′,5-tri-iodo-l-thyronine transport by human erythrocytes and by `ghosts'' prepared from these cells. Uptake of tri-iodothyronine by erythrocytes at 37°C was time-dependent with a maximum reached after 60min. Tracer analysis after incubation for 1min revealed only one saturable binding site, with K<sub>m</sub> 128±19nm (mean±s.e.m.; n=7) and V<sub>max.</sub> 4.6±0.7pmol of tri-iodothyronine/min per 6×10<sup>7</sup> cells. After 10min incubation K<sub>m</sub> 100±16nm (n=10) was found with V<sub>max.</sub> 7.7±1.2pmol of tri-iodothyronine/10min per 6×10<sup>7</sup> cells. At 0°C the uptake system is still active, with K<sub>m</sub> 132±26nm and V<sub>max.</sub> 1.8±0.3pmol of tri-iodothyronine/10min per 6×10<sup>7</sup> cells. The V<sub>max.</sub> with intact cells is 5-fold greater than the V<sub>max.</sub> with membranes derived from the same amount of cells when uptake studies are performed in media with similar free tri-iodothyronine concentrations. This indicates that at least 80% of tri-iodothyronine taken up by the intact erythrocytes enters the cell. This saturable uptake system can be inhibited by X-ray-contrast agents in a dose-dependent fashion. (±)-Propranolol, but not atenolol, has the same effect, indicating that the membrane-stabilizing properties of (±)-propranolol are involved. Furthermore, there is no inhibition by ouabain or vanadate, which indicates that tri-iodothyronine uptake is not dependent on the activity of Na<sup>+</sup>+K<sup>+</sup>-dependent adenosine triphosphatase. We have prepared erythrocyte `ghosts'', resealed after 2.5min with 0mm-, 2mm- or 4mm-ATP inside. Inclusion of ATP and integrity of the membrane of the erythrocyte `ghosts'' were verified on the basis of an ATP-concentration-dependent functioning of the Ca<sup>2+</sup> pump. No difference was found in the uptake of tri-iodothyronine by erythrocyte `ghosts'' with or without ATP included, indicating that uptake of tri-iodothyronine is not ATP-dependent. The following conclusions are drawn. (1) Tri-iodothyronine enters human erythrocytes. (2) There is only one saturable uptake system present for tri-iodothyronine, which is neither energy (i.e. ATP)-dependent nor influenced by the absence of an Na⁺ gradient across the plasma membrane. This mode of uptake of tri-iodothyronine by human erythrocytes is in sharp contrast with that of rat hepatocytes, which uptake system is energy-dependent and ouabain-sensitive Krenning, Docter, Bernard, Visser & Hennemann (1978) FEBS Lett. 91, 113–116; Krenning, Docter, Bernard, Visser & Hennemann (1980) FEBS Lett. 119, 279–282]. (3) X-ray-contrast agents inhibit tri-iodothyronine uptake by erythrocytes in a similar fashion to that by which they inhibit the uptake of tri-iodothyronine by rat hepatocytes Krenning, Docter, Bernard, Visser & Hennemann (1982) FEBS Lett. 140, 229–233].