Characterization of l-Thyroxine Transport into Hepatocytes Isolated from Juvenile Rainbow Trout (Oncorhynchus mykiss)

Abstract

Uptake of l-thyroxine (T4) by isolated trout hepatocytes was characterized after 1-min incubation of cells in a Hanks' balanced salts medium containing glucose and [125I]T4. Centrifugation through silicone oil and glycine buffer (pH 10.5) was used to separate cells from the medium and minimize extracellular binding of T4. At a T4 concentration of 0.2 nM, 15% of hepatocyte uptake was due to nonsaturable (passive) diffusion. Saturable T4 uptake obeyed Michaelis-Menten kinetics with an apparent Kt of 0.52 μM and a Jmax of 120 pmol/min/106 cells at 12°. T4 uptake was temperature-dependent. The activation energy was 58.0 kJ/mol between 1 and 6°, and 22.7 kJ/mol between 12 and 24°. T4 uptake was enhanced by increased H+ concentration over the range of 6.0-9.0 pH units and decreased sharply below pH 5.0. Dinitrophenol (1 mM) and potassium cyanide (2 mM) decreased T4 uptake to, respectively, 91 and 66% of controls, indicating a requirement for metabolic energy. The sodium ionophore monensin (10 μM) and the Na+ -H+ exchange inhibitor amiloride (0.5 mM) reduced uptake to 81 and 74%, respectively, of controls, while preincubation with ouabain (0.5 mM) or substitution of LiCl for NaCl in the medium did not modify T4 uptake, indicating that T4 transport does not depend on the presence of external Na+. The sulfhydryl-blocking reagent p-hydroxymercuribenzoate (0.5 mM) depressed T4 uptake to 32% of controls, but N -ethylmaleimide (0.5 mM) was ineffective. Inhibitors of protein binding, bromosulphothalein (0.3 mM) and 8-anilino-1-naphthalene sulfonic acid (1 mM), both decreased T4 uptake to 32 and 36% of controls, respectively, while phloretin (0.1 mM) and 5,5′-diphenylhydantoin, specific inhibitors of carrier-mediated transport, depressed uptake to 8 and 32% of controls, respectively, 3,5,3′-triiodo-l-thyronine (T3) decreased T4 uptake with an apparent Ki of 0.19 μM, indicating that T3 and T4 may share a common transport site(s). Other structural analogues, tested at 0.37 μM, influenced uptake as follows: 3,3′,5′-triiodo-l-thyronine (rT3) to 76% of control; 3,5-diiodo-l-thyronine to 57%; d-thyroxine to 93%; and 3,5,3′-triiodothyropropionic acid to 78%. l-tyrosine and l -phenylalanine (each at 0.2 mM) did not decrease T4 uptake. Colchicine (0.5 mM), an inhibitor of endocytosis, decreased T4 uptake to 75% of control when added with T4 and to 53% when added to cells 3 min prior to addition of T4. We conclude that T4 uptake into trout hepatocytes is a saturable, stereospecific, energy-dependent carrier-mediated transport that does not depend on extracellular Na+ but may involve an endocytotic mechanism. T3 and some other structural analogues of T4 may also be transported by this system.