oligomycins and monodansylcadaverine

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.

Topics: Amino Acids; Amino Acids, Cyclic; Analysis of Variance; Animals; Animals, Newborn; Biological Transport; Cadaverine; Cell Membrane; Cell Nucleus; Cells, Cultured; Cytosol; Diiodothyronines; Endocytosis; Female; Male; Models, Animal; Myocardium; Oligomycins; Rats; Rats, Wistar; Regression Analysis; Thyroxine; Triiodothyronine; Triiodothyronine, Reverse; Tryptophan; Tyrosine

The uptake of [125I]T4 was investigated in cultured anterior pituitary cells isolated from adult fed Wistar rats and cultured for 3 days in medium containing 10% fetal calf serum. Experiments were performed with [125I]T4 (10(5) to 2 x 10(6) cpm; 0.35-7 nM) in medium containing 0.5% or 0.1% BSA. The uptake of [125I]T4 increased with time and showed equilibrium after around 1 h of incubation. The presence of 10 microM unlabeled T4 during incubation decreased the uptake of [125I]T4 by 65-70% at all time intervals. After 24 h of incubation, 1.5% iodide and 3.2% conjugates were detected in the medium, whereas around 20% of cellular radioactivity represented [125I]T3. The 15-min uptake of [125I]T4 was significantly reduced by simultaneous incubation with 100 nM T4 (by 24%; P < 0.05), 100 nM T3 (by 38%; P < 0.001), or 10 microM rT3 (by 32%; P < 0.001), whereas 10 microM tetraiodothyroacetic acid (Tetrac) had no effect. Furthermore, preincubation (30 min) and incubation (15 min) with 10 microM monodansylcadaverine, oligomycin, or monensin reduced the uptake of [125I]T4 by 30%, 50%, and 40%, respectively (all P < 0.001). Substitution of Na+ in the buffer by K+ diminished the uptake of [125I]T4 by 39% (P < 0.005); 2 mM phenylalanine, tyrosine, or tryptophan reduced [125I]T4 uptake by 18% (P < 0.05), 18% (P = NS), and 33% (P < 0.005), respectively. Our data suggest that the pituitary contains a specific carrier-mediated energy-requiring mechanism for [125I]T4 uptake that is partly dependent on the Na+ gradient. In addition, part of [125I]T4 uptake in the pituitary might occur through an amino acid transport system. When expressed per pM of free hormone, the 15-min uptake of [125I]T4 was approximately as high as that of [125I]T3. Because the reduction of [125I]T4 uptake by T4, T3, monodansylcadaverine, oligomycin, and monensin was roughly the same as the previously reported reduction of [125I]T3 uptake by the same compounds, it is further suggested that T4 and T3 share a common carrier in cultured anterior pituitary cells.

Topics: Animals; Binding, Competitive; Cadaverine; Cell Membrane; Cells, Cultured; Iodine Radioisotopes; Kinetics; Male; Monensin; Oligomycins; Pituitary Gland, Anterior; Rats; Rats, Wistar; Serum Albumin, Bovine; Thyroxine; Triiodothyronine; Triiodothyronine, Reverse

Thyroid hormone entry into the thymocyte, a thyroid hormone target, was investigated by incubating the cells with tracer amounts of [125I]L-T3. At 37 C T3 uptake was linear with time up to 2 min, and then approached a plateau. The specific T3 uptake, obtained by subtracting the uptake in the presence of excess unlabeled T3, represented 48 +/- 6% of the total at equilibrium. Unlabeled L-T4, D-T3, and triiodothyroacetic acid were less effective than L-T3 in reducing [125I]T3 uptake. Kinetic studies on the initial rate of T3 uptake indicated, for the saturable process, a maximum velocity of approximately 1 pmol/10(6) cells.min and a Km of approximately 0.8 nM. Lowering incubation temperature to 4 C resulted in a two thirds reduction of the total T3 uptake. Washout experiments indicated a different hormone release, being more rapid for cells incubated at 4 C than at 37 C; at 30 min 70% of labeled T3 was released when incubation was carried out at 4 C compared to only 35% after incubation at 37 C, indicating the major intracellular location of the hormone at the latter temperature. An energy requirement of T3 uptake in thymocytes was shown by sensitivity to oligomycin; the effect was dose dependent, showing a maximal decrease in specific uptake of 85%. The involvement of cation movement in the entry process of T3 was indicated by the sensitivity to ouabain. These results indicate the existence of a stereospecific, energy-dependent, saturable process for T3 entry in thymocytes.

Topics: Animals; Cadaverine; Carrier Proteins; Intracellular Membranes; Iodine Radioisotopes; Mice; Oligomycins; Osmolar Concentration; Ouabain; Temperature; Thymus Gland; Time Factors; Triiodothyronine

The mechanism of T3 entry into cells was studied in undifferentiated NB41A3 neuroblasts and after differentiation with 0.5 mM sodium butyrate. In undifferentiated neuroblasts, cell uptake of labeled L-T3 at 2 h was reduced to 43% of the control value by excess L-T3, whereas only 5.9% of D-T3 uptake was saturable. After incubation of intact cells with labeled hormone, the nuclei contained 3.8% of the total cellular L-T3 and 4.3% of D-T3. Thus, L-T3 nuclear uptake was 3 times higher than D-T3. Kinetic analysis of the initial rate of uptake of L-T3 by the cells gave a Km of 1.25 nM and a maximum velocity of 2.38 fmol/min.10(6) cells. The initial rate of D-T3 uptake was not saturable. Inhibitors of ATP production (antimycin and oligomycin) as well as monodansylcadaverine virtually abolished saturable cell uptake and decreased nuclear uptake more than total cell uptake, suggesting that the saturable component of uptake into the cells is the major source of nuclear L-T3. After differentiation by butyrate, both cell uptake and nuclear uptake of L-T3 increased. The apparent affinity (Ka) of the nuclear T3 receptors was determined in intact cells and compared with the Ka measured with isolated nuclei. In undifferentiated cells, the apparent Ka was 3-fold higher than the true Ka, presumably due to a step-up in the free T3 concentration in the cytosol compared to that in the incubation medium. After butyrate exposure, the apparent nuclear Ka was decreased to less than 1.5 times the control value, but Ka in isolated nuclei was unchanged. The number of nuclear receptors, however, was increased by butyrate in both intact cell and isolated nuclei experiments. These results indicate that butyrate exerts separate effects on the number of nuclear receptors and saturable T3 transport in mouse neuroblasts.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Biological Transport; Butyrates; Butyric Acid; Cadaverine; Cell Differentiation; Cell Nucleus; Cytosol; Glucose; Kinetics; Mice; Neuroblastoma; Oligomycins; Stereoisomerism; Triiodothyronine; Tumor Cells, Cultured

The mechanism(s) responsible for the different biological potency of L- and D-T3 was investigated in rat L6E9 myoblasts. After incubation with intact cells at 37 C L-T3 cellular and nuclear uptakes were 91% and 70% higher than those of D-T3, respectively, but values for nuclear uptake as a fraction of cellular uptake were similar. The difference between the enantiomers was abolished at 4 C, and metabolic and endocytotic inhibitors reduced nuclear and extranuclear saturable uptake of L-T3 to a similar degree, but had little or no effect on D-T3 uptake. The affinity constants (Ka) for L- and D-T3 binding to isolated nuclei were similar, but the apparent nuclear Ka of L-T3 in intact cells was 5-fold greater than that of D-T3. The findings suggest that stereospecific transport, mainly active at the plasma membrane, occurs in rat skeletal muscle cells. This discriminative pathway of cell entry facilitates L-T3 uptake by an energy-dependent pathway not shared by D-T3 and may explain the greater potency of L-T3 than D-T3.

Topics: Animals; Antimycin A; Biological Transport; Cadaverine; Cell Line; Cell Membrane; Cell Nucleus; Iodine Radioisotopes; Kinetics; Muscles; Oligomycins; Rats; Stereoisomerism; Triiodothyronine

The uptake of [125I]T3 in rat skeletal muscle was investigated by incubating intact soleus muscles with a tracer amount of [125I]T3. At 37 C [125I]T3 uptake increased asymptotically; at 60 min the muscle contained 10% of the total [125I]T3 or 0.238 +/- 0.021% per mg wet tissue. At 0 C the [125I]T3 uptake was 1/5 of that at 37 C. The specific [125I]T3 uptake, determined by subtracting the uptake in the presence of 10 microM unlabeled T3 from the total [125I]T3 uptake, attained a plateau after 60 min. Washout experiments, done by first incubating the muscle for 60 min at 37 C or 0 C with [125I]T3 and then at 0 C for 3 h with unlabeled T3, showed that 21 +/- 2% or 58 +/- 4% of the radioactivity, respectively, was released, indicating an intracellular location of the hormone after incubation at 37 C. Addition of increasing concentrations of L-T3, D-T3 and L-T4 caused a progressive inhibition of the [125I]T3 uptake; the 50% inhibitory concentrations being 400 nM, 7 microM, and more than 15 microM, respectively. Preincubation of soleus muscles with metabolic inhibitors almost completely inhibited [125I]T3 specific uptake, with oligomycin and antimycin causing 98 +/- 4% and 81 +/- 3% reduction, respectively. Monodansylcadaverine and bacitracin, inhibitors of receptor-mediated endocytosis, reduced the specific [125I]T3 uptake in a dose-dependent manner up to 67 +/- 3% and 62 +/- 2%, respectively. These results indicate the presence of a saturable, stereospecific, and energy-dependent process responsible, at least in part, for T3 uptake in rat skeletal muscle. This specific T3 uptake may be a receptor-mediated endocytosis process.

Topics: Animals; Antimycin A; Bacitracin; Biological Transport, Active; Cadaverine; Endocytosis; In Vitro Techniques; Male; Muscles; Oligomycins; Rats; Temperature; Triiodothyronine