Search Results

You are looking at 1 - 2 of 2 items for

  • Author: N Shimada x
  • Refine by Access: All content x
Clear All Modify Search
Free access

N Shimada and K Yamauchi

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.

Free access

M Shimada, J Ito, Y Yamashita, T Okazaki, and N Isobe

In this study, we investigated the mechanisms of protein kinase B (PKB) activation and its role in cumulus cells during in vitro meiotic resumption of porcine oocytes. PKB activity in cumulus cells was significantly decreased by 12 h cultivation of cumulus-oocyte complexes (COCs) in basic medium. However, the addition of phosphodiesterase inhibitors, hypoxanthine or 3-isobutyl-1-methylxanthine, maintained the level of PKB activity in cumulus cells at comparable with that in cumulus cells just after collection from their follicles. When COCs were cultured with phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, PKB activity was significantly decreased, and both caspase 3 activity and the proportion of apoptotic cells were significantly increased as compared with those in cumulus cells just after collection from their follicles. Moreover, the inhibitory effect of hypoxanthine on spontaneous meiotic resumption was overcome by addition of LY294002. On the other hand, markedly high activity of PKB and high intensity of the phosphorylated PKB band were observed in cumulus cells of COCs which were cultured with FSH. The addition of 20 microM LY294002 to FSH-containing medium induced an apoptosis of cumulus cells, whereas little apoptotic-positive signal was detected in COCs cultured with 5 microM LY294002 and FSH. However, the inhibitory effects of LY294002 on progesterone production by cumulus cells and germinal vesicle breakdown in oocytes reached a maximum at 5 microM. Thus, high activity of the PI 3-kinase-PKB pathway in cumulus cells plays an important role in FSH regulation of cell function. Judging from these results, it is estimated that PI 3-kinase in cumulus cells is required for both the suppression of spontaneous meiotic resumption and the induction of gonadotropin-stimulated meiotic resumption.