We characterized the 3,5,3′-L-triiodothyronine (T3)- uptake system on the plasma membrane of Rana catesbeiana tadpole red blood cells (RBCs) in the presence of a variety of inhibitors and potentially competing amino acids. Saturable [125I]T3 uptake was inhibited by phloretin, monodansylcadaverine, bromosulfophthalein, sodium taurocholate and tryptophan. Saturable uptake obeyed simple Michaelis–Menten kinetics with an apparent K m of 110 nM and a V max of 2.5 pmol/min per 106 cells at 23 °C. These results suggested that a large proportion of T3 transported into RBCs was mediated by the aromatic amino acid transporter (System T)-linked transporter. To investigate the effect of endocrine-disrupting chemicals (EDCs) on [125I]T3 uptake, RBCs were incubated with [125I]T3 in the presence of each chemical. Among the test chemicals, di-n-butyl phthalate, n-butylbenzyl phthalate and the miticide, dicofol, were the most powerful inhibitors of [125I]T3 uptake, with an IC50 of 2.2 μM, which was one order of magnitude greater than that for T3 (IC50, 0.14 μM), and diethylstilbestrol and ethinylestradiol were modest inhibitors. Tributyltin accelerated saturable initial [125I]T3 uptake by 2-fold at 3.2 μM. When RBCs were cultured with 10 nM T3 at 25 °C for 2 days in the presence of monodansylcadaverine, ethinylestradiol, ioxynil or dicofol at the defined concentrations, these compounds inhibited significantly the induction of the thyroid hormone receptor α gene by T3. However, not all chemicals competed with T3 binding to the receptor at the same concentrations. Our results raise the possibility that the T3-uptake system on the plasma membrane of the tadpole RBCs could be a candidate target site for some EDCs and can modulate cellular T3 response.
K. Yamauchi, R. Horiuchi and H. Takikawa
The mechanisms of 3,5,3′-l-tri-iodothyronine (T3) uptake into human erythrocytes were examined. Purified membranes of human erythrocytes were shown to have two classes of T3-binding sites with one being a high-affinity site (dissociation constant, 59·2±17·8 nmol/l; maximum binding capacity, 344·3 ± 95·5 fmol/μg protein). Furthermore, it was shown that there were two pathways for T3 uptake in human erythrocytes; one was saturable, stereospecific (T3»thyroxine > 3,5,3′-d-tri-iodothyronine), energydependent and dominant at 15 °C; the other was not displaced by unlabelled T3 and was energyindependent but did not occur by passive diffusion. The former pathway which, it is suggested, is a receptor-mediated transport pathway, was inhibited by monodansylcadaverine, phloretin or oligomycin at 15 or 37 °C, but the latter pathway was not inhibited by these inhibitors. Our results strongly suggest that uptake of T3 by the energy-independent pathway became predominant over the energy-dependent pathway at 37 °C and accounted for 83% of total T3 uptake of human erythrocytes.
Journal of Endocrinology (1989) 121, 585–591
A. Sakurai, K. Ichikawa, K. Hashizume, T. Miyamoto, K. Yamauchi, H. Ohtsuka, Y. Nishii and T. Yamada
The effects of histone subfractions on rat liver thyroid hormone receptor–DNA interaction were examined using an in-vitro DNA-cellulose binding assay. H1 histones bound to DNA showed reversible and potent inhibition of receptor–DNA binding without affecting receptor–hormone binding. Poly-lysine, bovine serum albumin, ovalbumin and cytochrome c did not alter receptor–DNA binding. H1 histone subfractions (calf thymus lysine-rich histone (CTL)-1, CTL-2 and CTL-3) showed potent inhibition of receptor–DNA binding indistinguishable from each other. The quantity of H1 histone subfractions bound to DNA was the same. Although each subfraction has different functional properties, inhibition of receptor–DNA binding was a common feature of all the H1 histone subfractions, which is important for the non-random distribution of the receptor in chromatin.
Binding of the receptor to core histones was investigated; it was found to bind to core histones more potently than to other proteins (H1 histone, ovalbumin and cytochrome c). Among core histone subfractions, H4 histone bound to the receptor most potently and is the candidate to be one of the acceptor sites of the receptor in chromatin.
Journal of Endocrinology (1989) 121, 337–341
K. Ichikawa, K. Hashizume, T. Miyamoto, Y. Nishii, K. Yamauchi, H. Ohtsuka and T. Yamada
An aqueous two-phase partitioning study of partially purified nuclear thyroid hormone receptor from rat liver was performed. Stability of 3,5,3′-tri-iodo-l-thyronine (T3)–receptor complex and T3-binding activity in the presence of dextran or polyethylene glycol were assessed in order to determine the amount of occupied or unoccupied receptors in each phase. Partition coefficients were calculated as the ratio of receptor concentration in the upper polyethylene glycol-rich phase H2O and that in the lower dextranrich phase H2O. The partition coefficient was a sensitive function of the salt at pH above 6·1 and below 5·1. The salt had no effect on the partition coefficient at pH around 5·6. These results suggest that the isoelectric point of the thyroid hormone receptor is about 5·6, confirming previous determinations using isoelectric focusing. The partition coefficient of the receptor decreased upon T3 binding, regardless of the salt composition. In contrast, the partition coefficient of thyroxine-binding globulin increased upon T3 binding. Free T3 preferentially partitioned into the upper polyethylene glycol-rich phase and gave a partition coefficient higher than 1·0. These results strongly suggest that the decrease in the partition coefficient of the receptor upon hormone binding reflects conformational changes or changes in electrostatic properties of the receptor upon hormone binding. Such an alteration may be involved in biological activation of the receptor upon hormone binding.
J. Endocr. (1988) 119, 431–437
Y. Nishii, K. Hashizume, K. Ichikawa, T. Miyamoto, S. Suzuki, T. Takeda, K. Yamauchi, M. Kobayashi and T. Yamada
Changes in the amount of cytosolic 3,5,3′-tri-iodo-l-thyronine (T3)-binding protein (CTBP) and its activator during administration of l-thyroxine (T4) to thyroidectomized rats were investigated. Thyroidectomy decreased the amount of CTBP in the kidney, whereas the activator was not significantly modified by thyroidectomy. The activator was increased by administration of T4 to thyroidectomized rats. The amount of CTBP was also increased by administration of T4. The activator increased the maximal binding capacity (MBC) without changes in the affinity constant for T3 binding in CTBP. A T4-induced increase in MBC in cytosol inhibited nuclear T3 binding in vitro by competition of T3 binding between CTBP and the nuclear receptor.
These results suggest that thyroid hormone increases the capacity for cytosolic T3 binding through increasing the amount of CTBP and its activator, and that these increases play a role in regulating the amount of T3 that binds to its nuclear receptor.
Journal of Endocrinology (1989) 123, 99–104
S Yamada, M Komatsu, T Aizawa, Y Sato, H Yajima, T Yada, S Hashiguchi, K Yamauchi and K Hashizume
When isolated rat pancreatic islets are treated with 16.7 mM glucose, a time-dependent potentiation (TDP) of insulin release occurs that can be detected by subsequent treatment with 50 mM KCl. It has been thought that TDP by glucose is a Ca2+-dependent phenomenon and only occurs when exposure to glucose is carried out in the presence of Ca2+. In contrast to this, we now demonstrate TDP under stringent Ca2+-free conditions (Ca2+-free buffer containing 1 mM EGTA). In fact, under these Ca2+-free conditions glucose caused an even stronger TDP than in the presence of Ca2+. TDP induced by glucose in the absence of extracellular Ca2+ was unaffected by inhibitors of protein kinase C (PKC). However, cerulenin or tunicamycin, two inhibitors of protein acylation, eradicated TDP without affecting glucose metabolism. The TDP by glucose was not associated with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) during subsequent treatment with high K+. Exposure of islets to forskolin under Ca(2+)-free conditions did not cause TDP despite a large increase in the cellular cAMP levels. In conclusion, glucose alone induces TDP under stringent Ca2+-free conditions when [Ca2+]i was significantly lowered. Protein acylation is implicated in the underlying mechanism of TDP.
T Aizawa, T Kaneko, H Yajima, S Yamada, Y Sato, Y Kanda, S Kanda, M Noda, T Kadowaki, M Nagai, K Yamauchi, M Komatsu and K Hashizume
Oscillation of insulin release by the pancreatic islets was evaluated under stringent Ca(2+)-free conditions for the first time. Isolated single rat islets were exposed to 16.7 mM glucose in the presence of 1.9 mM Ca(2+), or under the stringent Ca(2+)-free conditions (Ca(2+) omission with 1 mM EGTA, 6 microM forskolin and 100 nM phorbol 12-myristate 13-acetate). Fifteen minutes after the initiation of glucose stimulation, effluent was collected at a 6-s interval, insulin was determined in duplicate by a highly sensitive insulin radioimmunoassay, and oscillation and pulsatility of release statistically analyzed. Significant oscillation of insulin release was observed in all islets irrespective of presence and absence of Ca(2+). Significant pulsatility of release was detected in 7 of 11 islets in the presence of Ca(2+) and three of six isl! ets in the absence of Ca(2+). In conclusion, high glucose elicits oscillatory insulin release both in the presence and absence of extracellular Ca(2+).