We have investigated the potential role of fibroblasts in local thyroid hormone metabolism in neonatal rat heart. Incubation of cardiac fibroblasts with thyroxine (T4) or 3,5,3'-tri-iodothyronine (T3) resulted in the appearance of water-soluble metabolites, whereas incubation of cardiomyocytes under the same conditions did not or did so to a much lesser extent. Time-course studies showed that production is already evident after 1-5 h of exposure and that the process equilibrates after 24-48 h. Analysis of the products revealed both the T4 and the T3 metabolites to be glucuronides. These results were corroborated by the detection of uridine diphosphate (UDP)-glucuronyltransferase activity in cardiac fibroblasts. We found no indication for outer ring deiodination in fibroblasts, cardiomyocytes or heart homogenates. From these results we have concluded that cardiac fibroblasts, but not cardiomyocytes, are able to glucuronidate T4 and T3 and secrete the conjugates. This could play a role in local metabolism, e.g. to protect the heart tissue from high levels of thyroid hormones.

N-TERA-2 cl/D1 (NT2) cells, a human embryonal cell line with characteristics of central nervous system precursor cells, were utilised to study thyroid hormone action during early neuronal growth and differentiation. Undifferentiated NT2 cells expressed mRNAs encoding thyroid hormone receptors (TRs) alpha1, alpha2 and beta1, iodothyronine deiodinases types 2 (D2) and 3 (D3) (which act as the pre-receptor regulators), and the thyroid hormone-responsive genes myelin basic protein (MBP) and neuroendocrine specific protein A (NSP-A). When terminally differentiated into post-mitotic neurons (hNT), TRalpha1 and TRbeta1 mRNA expression was decreased by 74% (P=0.05) and 95% (P<0.0001) respectively, while NSP-A mRNA increased 7-fold (P<0.05). However, mRNAs encoding TRalpha2, D2, D3 and MBP did not alter significantly upon neuronal differentiation and neither did activities of D2 and D3. With increasing 3,5,3'-triiodothyronine (T(3)) concentrations, TRbeta1 mRNA expression in cultured NT2 cells increased 2-fold at 10 nM T(3) and 1.3-fold at 100 nM T(3) (P<0.05) compared with that in T(3)-free media but no change was seen with T(3) treatment of hNT cells. D3 mRNA expression in NT2 cells also increased 3-fold at 10 nM T(3) (P=0.01) and 2.4-fold at 100 nM T(3) (P<0.05) compared with control, but there was no change in D3 enzyme activity. In contrast there was a 20% reduction in D3 mRNA expression in hNT cells at 10 nM T(3) (P<0.05) compared with control, with accompanying reductions in D3 activity with increasing T(3) concentrations (P<0.05). There was no significant change in the expression of the TRalpha isoforms, D2, MBP and NSP-A with increasing T(3) concentrations in either NT2 or hNT cells. Undifferentiated NT2 and differentiated hNT cells show differing patterns of T(3)-responsiveness, suggesting that there are different regulatory factors operating within these cell types.

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.