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

Abstract

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 degrees C was time-dependent with a maximum reached after 60min. Tracer analysis after incubation for 1min revealed only one saturable binding site, with K(m) 128+/-19nm (mean+/-s.e.m.; n=7) and V(max.) 4.6+/-0.7pmol of tri-iodothyronine/min per 6x10(7) cells. After 10min incubation K(m) 100+/-16nm (n=10) was found with V(max.) 7.7+/-1.2pmol of tri-iodothyronine/10min per 6x10(7) cells. At 0 degrees C the uptake system is still active, with K(m) 132+/-26nm and V(max.) 1.8+/-0.3pmol of tri-iodothyronine/10min per 6x10(7) cells. The V(max.) with intact cells is 5-fold greater than the V(max.) 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(+)+K(+)-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(2+) 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].