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The uptake of tri-iodothyronine (T(3)) in cultured neonatal rat cardiomyocytes was investigated and compared with the uptake of reverse T(3 )(rT(3)) and thyroxine (T(4)). Cellular compartmentalization of T(3) was studied by distinguishing T(3) activity associated with the plasma membrane from that in the cytosol or incorporated in the cell nucleus. T(3) and T(4) uptake displayed similar temperature dependencies which, in magnitude, differed from that of rT(3) uptake. T(3) uptake was Na(+ )independent, and sensitive to oligomycin and monodansylcadaverine (42-49% and 25% inhibition of 15-min cellular uptake respectively). Furthermore, T(3) uptake could be inhibited by tryptophan (20%) and tyrosine (12%), while 2-aminobicyclo[2,2,1]heptane-carboxylic acid had no effect. Co-incubation with tryptophan and oligomycin resulted in an additive inhibition of T(3) uptake (77%). We therefore conclude that (i) T(3) uptake is energy dependent, (ii) receptor-mediated endocytosis may be involved and (iii) the aromatic amino acid transport system T may play a role, while system L is not involved in T(3) transport in cardiomyocytes. Co-incubation with unlabeled iodothyronines showed that 3,3'-di-iodothyronine and T(3) itself were the most effective inhibitors of T(3) uptake (30% and 36% inhibition of 15-min cellular uptake respectively). At 15-min incubation time, 38% of the total cell-associated T(3) was present in the cytosol and nucleus, and 62% remained associated to the plasma membrane. Unidirectional uptake rates did not saturate over a free T(3) concentration range up to 3.9 microM. We have concluded that T(3) uptake in neonatal rat cardiomyocytes occurs by an energy- and temperature-dependent mechanism that may include endocytosis and amino acid transport system T, and is not sensitive to the Na(+) gradient. Elucidation of the molecular basis for the T(3) transporter is the subject of current investigation.

Uptake of tri-iodothyronine (T(3)) was compared with that of thyroxine (T(4)) in the embryonic heart cell line H9c2 (2-1). These cells propagate as myoblasts and form differentiated myotubes upon reduction of the serum concentration, as indicated by a 31-fold increase in creatine kinase activity. Protein and DNA content per well were around 2-fold higher in myotubes than in myoblasts. When expressed per well, T(3) and T(4) uptake were, compared with myoblasts, 1.9- to 2-fold and 3.1- to 4-fold higher in myotubes respectively. On the other hand, the characteristics of T(3) and T(4) uptake were similar in myoblasts and myotubes. At any time-point, T(4) uptake was 2-fold higher than that of T(3), and both uptakes were energy but not Na(+) dependent. T(3) and T(4) uptake exhibited mutual inhibition in myoblasts and myotubes: 10 microM unlabeled T(3) reduced T(4) uptake by 51-60% (P<0.001), while 10 microM T(4) inhibited T(3) uptake by 48-51% (P<0.001). Furthermore, T(3) and T(4) uptake in myoblasts was dose-dependently inhibited by tryptophan (maximum inhibition around 70%; P<0.001). Exposure of the cells to T(3) or T(4) during differentiation significantly increased the fusion index (35 and 40%; P < 0.01). Finally, both myoblasts and myotubes showed a small deiodinase type I activity, while deiodinase type II activity was undetectable. In conclusion, T(3) and T(4) share a common energy-dependent transport system in H9c2(2-1) cells, that may be important for the availability of thyroid hormone during differentiation.

Cellular and nuclear uptake of [125I]tri-iodothyronine (T3) and [125I]triiodothyroacetic acid (Triac) were compared in cardiomyocytes of 2-3 day old rats, and the effect of thyroid hormone analogs on cellular T(3) uptake was measured. Cells (5-10 x 10(5) per well) were cultured in DMEM-M199 with 5% horse serum and 5% FCS. Incubations were performed for from 15 min to 24 h at 37 degrees C in the same medium, 0.5% BSA and [125I]T3 (100 pM), or [125I]Triac (240 pM). Expressed as % dose, T(3) uptake was five times Triac uptake, but expressed as fmol/pM free hormone, Triac uptake was at least 30% (P<0.001) greater than T3 uptake, whereas the relative nuclear binding of the two tracers was comparable. The 15 min uptake of [125I]T3 was competitively inhibited by 10 microM unlabeled T3 (45-52%; P<0.001) or 3,3'- diiodothyronine (T2) (52%; P<0.001), and to a smaller extent by thyroxine (T(4)) (27%; 0.05<0.1). In contrast, 10 microM 3,5-T2, Triac, or tetraiodothyroacetic acid (Tetrac) did not affect T3 uptake after 15 min or after 24 h. Diiodothyropropionic acid (DITPA) (10 microM) reduced 15-min T3 uptake by about 24% (P<0.05), but it had a greater effect after 4 h (56%; P<0.001). Exposure to 10 nM DITPA during culture reduced cellular T3 uptake, as did 10 nM T3, suggesting down-regulation of the plasma membrane T3 transporters. We conclude that i) Triac is taken up by cardiomyocytes; ii) 3,3'-T2 and, to a lesser extent, DITPA and T4 interfere with plasma membrane transport of T3, whereas 3,5-T2, Triac, or Tetrac do not; iii) the transport mechanism for Triac is probably different from that for T3.