title: Inhibition of uptake of thyroid hormone into rat hepatocytes by preincubation with N-bromoacetyl-3,3',5-triiodothyronine.
abstract: To investigate whether affinity coupling of N-bromoacetyl-T3 (BrAcT3) to the T3 membrane carrier results in an inhibition of transport of T3 into the cell, rat hepatocytes in monolayer were incubated for 2 h at 21 C with 1.3 mumol/liter BrAcT3 in medium without protein. After extensive washing, cells were incubated during 20 h at 37 C with [125I]T3 in medium with 0.5% BSA, and products in supernatants were analyzed by LH-20 column chromatography. In addition the apparent affinity constant (Km) and maximal uptake velocity (Vmax) of the high affinity uptake process were estimated using 1 min incubations of hepatocytes with various concentrations of T3. In control experiments (i.e. without BrAcT3 affinity coupling) about 57% of the added T3 was cleared from the medium and further metabolized, 85% of the cleared T3 reappeared in the medium as I-, 15% as conjugates. Addition of propylthiouracil during the 20 h incubation with T3 strongly inhibited deiodination, without a change in T3 clearance. Because T3 is sulfated before deiodination, a concomitant rise in conjugates was observed. Addition of ouabain to control cells during the 20 h incubation with T3 strongly inhibited uptake, with a parallel decrease in I- and conjugate formation. After affinity coupling of BrAcT3, T3 clearance was inhibited (by 30% P less than 0.001). Since I- production was more depressed (by 73%) than T3 clearance, with some rise in conjugate formation (P less than 0.001), inhibition of deiodinase by BrAcT3 also took place. The effects of BrAcT3 and ouabain on uptake of T3 appeared to be additive as were the effects of propylthiouracil and BrAcT3 on deiodination. After affinity coupling of BrAcT3, the Km of T3 uptake did not change significantly; however Vmax was 54% lower (P less than 0.025) indicating a noncompetitive inhibition of the transport system. Preincubation of the cells with N-acetyl-T3 does not alter the characteristics of uptake of T3 by rat hepatocytes as compared to controls, indicating that no binding of this compound occurs. It is concluded that preincubation of hepatocytes with BrAcT3 diminished I- formation from T3; 50% of this inhibition is due to decreased membrane transport and 50% by reduction of deiodination. Inhibition of membrane transport by BrAcT3 is substantiated by a 54% lower Vmax without a significant change in Km as compared to control. The effect of transport of thyroid hormone on metabolism stresses the importance of the membrane carrier in the translocation process.
authors.affiliation: Department of Internal Medicine III and Clinical Endocrinology, Erasmus University, Rotterdam, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Doctor
authors.forename: E P
authors.initials: EP
authors.lastname: Krenning
authors.forename: H F
authors.initials: HF
authors.lastname: Bernard
authors.forename: T J
authors.initials: TJ
authors.lastname: Visser
authors.forename: G
authors.initials: G
authors.lastname: Hennemann
mesh_terms: D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010042:Ouabain / Q000494:pharmacology; D011441:Propylthiouracil / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D014284:Triiodothyronine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors* / Q000494:pharmacology
chemical_list: D000345:Affinity Labels; D007454:Iodides; D014284:Triiodothyronine; D010042:Ouabain; D011441:Propylthiouracil; C036219:N-bromoacetyl-3,3',5-triiodothyronine