DRUG COMPETITION FOR INTRACELLULAR TRIIODOTHYRONINE-BINDING SITES

A variety of substances, including frusemide, non-esterified fatty aci ds (NEFAs) and non-steroidal antiinflammatory drugs (NSAIDs), can comp ete for triiodothyronine (T-3)-binding sites in serum and at the cell surface. We examined the competitive potency of these agents at intrac ellular T-3-binding sites in order to assess their potential to act as T-3 antagonists. Competition for [I-125]T-3 binding was determined us ing hydroxyapatite separation in cytosols and nuclear extracts prepare d from livers of Macaca fascicularis. The T-3 affinities were 15.8 +/- 1.2 nmol/l in cytosol and 0.23 +/- 0.02 nmol/l in nuclear extract. Do se-response curves were analysed by a four-parameter sigmoid curve-fit ting program to determine competitor potency. The nineteen agents test ed included various NSAIDs, NEFAs, non-bile acid cholephils (NBACs), f rusemide, amiodarone and the flavonoid EMD 21388. In nuclear extract t he most active competitors were linoleic acid (8.5 mu mol/l) and linol enic acid (7.8 mu mol/l). Potencies of NSAIDs varied between 66 mu mol /l (meclofenamic acid) and 525 mu mol/l (diclofenac). In cytosol, NEFA s were less potent but NSAIDs were stronger competitors than in nuclea r extract. Half-inhibitory potencies in cytosol were between 13.2 mu m ol/l (meclofenamic acid) and 63.1 mu mol/l (flufenamic acid). The NBAC bromosulphthalein was one of the most potent inhibitors in both cytos ol and nuclear extract. When expressed relative to T-3, diclofenac was a more effective competitor in cytosol than it was in nuclear extract . Amiodarone and EMD 21388 were without effect both in cytosol and nuc lear extract. Frusemide (759 mu mol/l) was weakly active in cytosol on ly. The action of Tg was assessed by measuring secretion of sex hormon e-binding globulin (SHBG) in Hep-G2 cells. After 3 days with total T-3 (0.1 mu mol/l), SHBG was 155 +/- 15% of the control. Amiodarone (100 mu mol/l) and meclofenamic acid (100 mu mol/l) were cytotoxic. Bromosu lphthalein (10 mu mol/l), one of the most potent competitors at both t he cytoplasmic and the nuclear level, did not influence the T-3-induce d rise in SHBG secretion. None of the drugs tested affected the magnit ude of maximal induction of SHBG by Tg. Substances that compete for se rum and cell surface T-3-binding sites are also weak competitors for i ntracellular T-3- binding proteins, although the hierarchy of potency differs. Frusemide and diclofenac, with a greater relative potency for cytosolic binding than nuclear binding, may have potential use in inv estigating the function of cytosolic T-3-binding. Amiodarone shows no binding activity and is not a hormone antagonist in primate hepatic ti ssue.