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.
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HH van der Putten, BJ Joosten, PH Klaren, and ME Everts
SM van der Heide, BJ Joosten, ME Everts, and PH Klaren
We have investigated the hypothesis that uridine 5'-diphosphate (UDP)-glucuronyltransferases (UGTs) and beta-glucuronidase are jointly involved in a mechanism for the storage and mobilization of iodothyronine metabolites in liver, kidney, heart and brain. Specifically, we predicted UGT activities to decrease and increase respectively, and beta-glucuronidase activity to increase and decrease respectively in hypo- and hyperthyroidism. To this end we have studied the effects of thyroid status on the activities of different enzymes involved in thyroid hormone metabolism in liver, kidney, heart and brain from adult rats with experimentally induced hypo- and hyperthyroidism. We used whole organ homogenates to determine the specific enzyme activities of phenol- and androsteron-UGT, beta-glucuronidase, as well as iodothyronine deiodinase types I and II. Deiodinase type I activities in liver and kidney were decreased in hypothyroid animals and, in liver only, increased in hyperthyroidism. Deiodinase type II activity was increased in hyperthyroid rat kidney only. Interestingly, in the heart, deiodinase type I-specific activity was increased fourfold, although the increase was not statistically significant. Cardiac deiodinase type I activity was detectable but not sensitive to thyroid status. Hepatic phenol-UGT as well as androsteron-UGT activities were decreased in hypothyroid rats, with specific androsteron-UGT activities two to three orders of magnitude lower than phenol-UGT activities. Both UGT isozymes were well above detection limits in heart, but appeared to be insensitive to thyroid status. In contrast, cardiac beta-glucuronidase activity decreased in hypothyroid tissue, whereas the activity of this enzyme in the other organs investigated did not change significantly.In summary, cardiac beta-glucuronidase, albeit in low levels, and hepatic phenol-UGT activities were responsive only to experimental hypothyroidism. Although a high basal activity of the pleiotropic beta-glucuronidase masking subtle activity changes in response to thyroid status cannot be ruled out, we conclude that hepatic, renal and cardiac UGT and beta-glucuronidase activities are not regulated reciprocally with thyroid status.
HH van der Putten, BJ Joosten, PH Klaren, and ME Everts
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.