OUDE Hypoforum

Endocrinology. First published ahead of print October 23, 2008 as doi:10.1210/en.2008-0627

Transport of thyroxine and 3,3’,5-triiodothyronine in human umbilical vein endothelial cells
Thyroid hormone transport in HUVECs

W. Edward Visser, Edith C.H. Friesema and Theo J. Visser
Department of Internal Medicine, Erasmus University Medical Center,
Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands

Figure legends
Fig. 1.
Time course (min) of uptake of 1 nM [125I]T3 (●) and [125I]T4 (○) in cultured HUVECs.
Uptake was measured in D-PBS containing 0.1% glucose and 0.1% BSA. Significances
represent differences between T3 and T4 uptake; * P < 0.05.

Fig. 2.
Effects of increasing concentrations (1 nM to 10 μM) unlabeled iodothyronines on the uptake
of [125I]T3 (A) in cultured HUVECs. Effects of 1 mM unlabeled aromatic amino acids (Phe,
Tyr, Trp) and BCH on the uptake of [125I]T3 (B) and [125I]T4 (C). Cells were incubated for 10
min. Net uptake in the presence of a competitor is expressed as a percentage of control uptake
(1 nM [125I]T3 or [125I]T4).

Fig. 3.
Na+ dependence of the uptake of [125I]T3 and [125I]T4 in cultured HUVECs. Cells were
incubated with 1 nM [125I]T3 or [125I]T4 for 10 min in sodium chloride medium (gray bars) or
in sodium-free choline (black bars) medium. Net uptake in the presence of choline is
expressed as a percentage of control (sodium chloride) uptake. * P < 0.05; ** P < 0.01.

Fig 4.
Effects of the Ca2+ channel blocker verapamil on T3 and T4 transport. Time course of the
effects of 0.1 mM verapamil on the uptake of 1 nM [125I]T3 (A) and [125I]T4 (B) in cultured
HUVECs. The efflux of 1 nM [125I]T3 (C) and [125I]T4 (D) in the presence of 0.1 mM
verapamil during the 30-min preincubation period (+/-, open triangle) or during the efflux
period only (-/+, filled triangle). Significances represent values compared to controls. * P <
0.05; ** P < 0.01; *** P < 0.001.
Fig. 5. (A) Affinity labeling of HUVECs. Intact cells were incubated for 4 h at 37 C with
BrAc[125I]T3. Subsequently, cells were harvested and cell lysates were separated by SDSPAGE.
Radioactivity was measured by phosphor imaging. (B) Expression of proteins, which
are associated with non-radiolabeled BrAcT3. Lysates of cells were separated by SDS-PAGE
and analyzed by Western blotting. BrAcT3-associated proteins were detected with a T3-
specific antibody. (C) Western blot on a lysate of HUVECs probed with a specific PDI
antibody. (D) Influence of treatment of cell lysates at pH 8 or 11 on the distribution of
BrAc[125I]T3-labeled proteins. Membranes were isolated and membrane proteins were
separated by SDS-PAGE. (E) Total radioactivity of BrAc[125I]T3-labeled proteins in
subcellular fractions after treatment of cell lysates at pH 8 or 11. * P < 0.01